The dierence in read length from that of 454 sequencing was compensated for by the increase of over two orders of magni tude inside the amount of reads. We demonstrated de novo assembly and analysis of the venom gland transcriptome working with only Illumina sequences and offered a compre hensive characterization of the two the toxin and nontoxin genes expressed in an actively producing snake venom gland. Outcomes and discussion Venom gland transcriptome sequencing and assembly We created a complete of 95,643,958 pairs of reads that passed the Illumina high-quality lter for 19 gigabases of sequence from a cDNA library with an average insert size of ?170 nt. Of those reads, 72,114,709 have been merged around the basis of their three overlap, yielding composite reads of average length 142 nt with normal phred attributes 40 as well as a total length 10 Gb.
This merging of reads lowered the eective selleck chemicals MK-2206 size of the data set without the need of loss of information and supplied lengthy reads to facilitate precise assembly. Our rst method to transcriptome assembly was aimed at identifying toxin genes. We attempted to implement as many of your information as you possibly can to make sure the identication of even the lowest abundance harmful toxins. To this end, we con ducted substantial searches of assembly parameter room for the two ABySS and Velvet around the basis on the full set of both merged and unmerged reads. We utilised the assemblies together with the most effective N50 values for more analysis. For Velvet, the assembly making use of a k mer size of 91 was very best. this assembly was subsequently analyzed with Oases.
For ABySS, the ideal k mer value was also read review 91, but due to the fact the overall performance with regards to total length transcripts appeared to depend strongly about the coverage and erode parameters, we further analyzed the k91 assemblies with c10 and e2, c100 and e100, and c1000 and e1000. We identied all total length harmful toxins by means of blastx searches within the benefits of all four assemblies. As a part of our rst technique, we also performed four independent de novo transcriptome assemblies with NGen 3 with twenty million merged reads each and every and one particular with the remaining 12,114,709 merged reads. We identied all full length harmful toxins from all 4 assemblies. Provided that all 3 assembly solutions tended to produce a big variety of fragmented toxin sequences, apparently because of retained introns and perhaps substitute splic ing, we designed and implemented a straightforward hash table technique to finishing partial transcripts, which we will refer to as Extender. We employed Extender on partial toxin sequences identied for two from the 4 NGen assemblies. We also annotated one of the most abundant total length nontoxin transcripts for the 3 assemblies based mostly on twenty million reads.
Moreover to its function as being a foods animal, the chicken includes a extended history as a beneficial model research organism. These dual considerations led to your variety of chicken since the 1st agricultural animal model to be sequenced with the gen ome level. While chickens happen to be used heavily for research of developmental biology and immunology, a num ber of traits make them a viable model for studies of adi pose biology, weight problems and insulin resistance. Industrial broiler chickens, in particular, rapidly accumulate excess adipose tissue as a result of genetic selection for growth and therefore are regarded as obese relative to leaner egg laying or wild strains of chickens. Chickens mimic the early stage of kind two diabetes in people, exhibiting both hyperglycemia and resistance to exogenous insulin.
Like people, but un like rodents or pigs, chickens depend upon liver instead of adi pose tissue for that majority of de novo lipid synthesis. Most metabolic genes are conserved with people, in addition to a quantity of the quantitative trait loci that have been MK-2206 Akt inhibitor linked to fatness in chickens contain genes implicated in human susceptibility to obesity or diabetes. Chickens also represent a model for studying mechanisms of adipo cyte hyperplasia through development, a course of action that could exacerbate grownup weight problems. During a minimum of the primary various weeks just after hatch, chicken adipose tissue expands more through adipocyte hyperplasia than hypertrophy, and an early enhance in adipocyte variety is really a widespread feature of some lines genetically picked for extra adiposity.
Lastly, the egg presents possibilities to straight selleck manipu late the developmental milieu and research the consequences on adipose metabolic process by way of in ovo injection. Relatively small is regarded about regulation of adipose tis sue deposition and metabolism in chicken. Because of its relative significance in lipogenesis, most research have fo cused about the function of liver in adipose growth. Several genetic lines of extra fat and lean chickens are created by means of phenotypic assortment, the majority of which have each ele vated plasma amounts of pretty reduced density lipoprotein and reduced levels of plasma glucose, reflecting the import ance of hepatic lipogenesis and glucose consumption in fat accretion. Reciprocally, phenotypic variety for very low plasma glucose concurrently selects for fatness.
Each chicken and mammalian adipocytes develop by means of a sequence of molecular triggers such as activation of CCAAT enhancer binding protein alpha and per oxisome proliferator activated receptor gamma. A clear point of divergence, on the other hand, is their respon siveness to insulin. In contrast to in mammals, insulin has min imal impact on glucose uptake in chicken adipose tissue. In reality, an avian homolog with the insulin delicate glu cose transporter GLUT4 has not been recognized in the latest chicken genome database.
Chondrocytes from three patients subjected to ACT, and another three individuals subjected to total knee arthroplasty, were in separate experiments challenged with 10 nM or 100 nM chemerin21 157 for 24 h, and subsequently a panel of cytokines was measured in the cell supernatants. The results show an increased concentration of TNF a, IL 1b, IL 6 and IL 8 as a result of chemerin stimulation in comparison to unstimulated control cells. The levels of IL 6 and IL 8 were markedly increased, whereas a rather modest effect was observed in terms of altered levels of IL 1b and TNF a. Joint inflammation is associated with deterioration of the cartilage matrix requiring a clarification as to whether chemerin21 157 affects chondrocyte secretion of matrix metalloproteases.
Cell cultures from six indivi duals were arranged and challenged with 10 nM or 100 nM chemerin21 157 for 24 h, and subsequently a panel of eight different MMPs was measured in the supernatants. Significantly elevated levels of MMP 1, MMP 2, MMP 3, MMP 8, and read the full info here MMP 13 were detected. The metalloproteases MMP 7, MMP 9, and MMP 12 could not be detected. Discussion Recent studies addressing the role of chondrocytes in joint inflammation have revealed that these cells secrete selleckchem TKI-258 and bind a variety of cytokines and chemokines and that they possess immunoregulatory cap abilities. The present study adds further informa tion to this issue by demonstrating that chondrocytes in both native cartilage and cell culture express the chemokine receptor ChemR23, a property primarily ascribed to leukocytes.
Using the ligand recombinant human chemerin21 157, we demonstrated that chemerinChemR23 binding eli cits intracellular signalling leading to the phosphoryla tion of p4442 MAPKs and Akt, both of which are involved in central signal transduction pathways that convey inflammatory signalling. Hence, the cleavage product of prochemerin chemerin21 157 mediates pro inflammatory signalling in chondrocytes as judged by the observed promotion of cytokine secretion. The enzymes reported to generate chemerin21 157 from prochemerin include the neutrophil serine pro teases cathepsin G and elastase. This indicates that, regardless of the source of prochemerin in joints, it can be cleaved by the enzymes produced by neutro phils into isoforms of chemerin that further promote inflammation by recruiting leukocytes, and that pro mote chondrocyte secretion of pro inflammatory cyto kines. Previous studies have reported that chemerin21 157 can be detected in arthritic synovial fluid and prochemerin from the circulation could likely be the source.
In conclusion, our data are steady with a model during which Th17 cells may participate in enhancing in flammation whilst concurrently limiting fibrosis. It’s really worth noting that the contribution of Th17 cells to inflam matory problems stays in lots of cases a matter of debate. As an example, the part of IL 17 while in the initiation, progression and stabilization of atherosclerosis is at the moment controversially interpreted with proof in favor of its proatherogenic possible and evidence in favor of its atheroprotective position. Our findings strain for your 1st time the concomitant dual purpose of Th17 cells in the context of matrix deposition and may perhaps supply the functional basis for novel approaches to harness fibrotic illnesses.
Conclusions Th17 cells improve in vitro fibroblast inflammatory responses although simultaneously inhibiting collagen produc tion having a mechanism partially dependent on IL 17, TNF and IFN. SSc fibroblasts are, nonetheless, intrinsically selleckchem resist ant to collagen inhibition induced by Th17 cells. Consequently, the elevated Th17 cell counts observed in SSc may be regarded a manifestation of autoimmunity not mechan istically linked to fibrosis. Introduction Articular chondrocytes undergo an apparent phenotypic alter when isolated from cartilage matrix and cultured in a monolayer. Through this alter, or dedifferentiation, the cell metabolic process of course adjustments, and also the matrix synthesized from the cells adjustments from 1 exclusive cartilage to a further much like that produced by fibroblasts.
Residing within cartilage matrix, chondrocytes express cartilage matrix components this kind of as form II collagen and aggrecan, but synthesize little variety I or style III procollagen, that are trace components of typical articular cartilage. With all the initiation of dedifferentiation, inhibitor mTOR inhibitors the expression of sort II collagen and aggrecan declines steadily, and also the expression of variety I and variety III procollagens is induced as a substitute. In parallel with this particular metabolic transform, the cell form improvements dramatically in the authentic spherical kind to flattened elongated varieties resembling those of fibroblasts. Despite the fact that dedifferentiation is really a important problem in tissue engineering, the precise mechanism for dedifferenti ation hasn’t been known for decades. In a latest examine, we reported that vB5 integrin may well perform a important part in dedifferentiation. In monolayer cultured chondrocytes, vB5 integrin suppresses the expression of cartilage matrix components by means of the activation of Elk related tyrosine kinase signaling, and promotes morphological modify from the cells. Even so, in that examine vB5 integrin was observed to not be concerned while in the induction of type I or style III procollagen expression.
To examine the expression of PTPN22 isoforms in macrophages, we quantified the transcript amount of PTPN22 isoforms in macrophages from 7 balanced donors. We uncovered that the ranges of Lyp2, PTPN22. 2, PTPN22. 56, PTPN22. 6, and PTPN22. 78 have been pretty comparable among resting, M1, and M2 macrophages. Consequently, the in crease in complete PTPN22 observed in M2 cells mainly comes from PTPN22. one. Taken collectively, our information indicate that the amount of PTPN22 isoforms varies considerably amongst cells styles and in response to diverse stimuli. Subcellular localization and perform of PTPN22 isoforms PTPN22 is made up of a NLS at its N terminus and it is also present from the nucleus of macrophage and T cells. This NLS is existing in all isoforms.
To even more examine the subcellular localization of PTPN22 isoforms, we expressed each isoform in 293 cells and separately ex amined the cytoplasmic and nuclear extract on the transfected cells with Western blotting. As anticipated, PTPN22. 1 was detected in both the cytoplasm and selleck chemical p38 inhibitors the nuclei from the transfected cells. A comparable pattern of subcellular localization was observed for Lyp2, PTPN22. two, and PTPN22. 5. Interestingly, we de tected PTPN22. 6 and PTPN22. 8 only inside the cytoplasm but not from the nucleus with the transfected cells, sugges ting the presence of an additional and crucial NLS encoded by exon six, that is spliced out in these two isoforms. PTPN22. 6 can act like a dominant unfavorable variant of PTPN22. one. But the function from the other isoforms is still unclear. We thus examined the result within the other isoforms on NFAT driven luciferase action.
As anticipated, overexpression of PTPN22. one in Jurkat cells suppressed experienced NFAT dependent luciferase activity by approximately 50%. Interestingly, Lyp2, PTPN22. 2, PTPN22. 5, and PTPN22. eight, in spite of missing elements in the PTP domain, also had the same effect. There was no statistically signi ficant distinction among these isoforms even after modify ment for your protein degree. Additionally, a catalytic dead mutant of mouse PTPN22, which has D195A and C227S mutations, had no effect on NFAT activity, more indicating that these isoforms are nevertheless catalytic active. In contrast, expression of PTPN22. six resulted in a subtle but statistically major grow in NFAT action. This consequence was reported before but was included for comparison.
Expression of PTPN22 isoforms in healthful and SLE populations To determine no matter whether the expression of PTPN22 iso forms was altered in SLE individuals and no matter whether the degree of PTPN22 isoforms was correlated with clinical capabilities of SLE, we quantified the transcript level of every isoform inside the peripheral blood of 15 healthful donors and 49 pa tients with SLE. The demographic qualities of the study topics are proven in Table 1. All healthful indi viduals have been female, but two within the 49 sufferers with lupus were male.
Nevertheless, there is certainly quite small evidence relating to the clinical benefit of IMRT for breast cancer. This distinctive NCRN adopted randomised controlled trial will test the clinical advantage of IMRT for females with early breast cancer. Strategies The main question is does correction of dose homogeneity applying forward planned IMRT boost the cosmetic outcome in sufferers with early breast cancerPatients with significant dose inhomogeneities with 2DRT are randomised to IMRT or regular 2D RT. High top quality normal tissue toxicity and cosmesis data are becoming collected, such as a novel analytical approach of breast volume measurement utilizing a 3D laser camera. Final results Eight hundred and eighty 5 sufferers happen to be recruited to date, and accrual of 1,000 sufferers is on target for January 2007.
A higher high-quality radiographer led 3D breast radiotherapy service has developed as a direct result in the trial. Blood DNA samples from trial sufferers will allow investigation of individual genetic variation in regular tissue selleckchem radiosensitivity within a multicentre translational radio genomics study. Conclusion The outcomes from this trial could provide impetus to improve the excellent of breast radiotherapy for many women worldwide. The DNA database will considerably contribute towards the ultimate aim of individualised radiotherapy primarily based on genetics. Breast Cancer Analysis 2006, eight P35 Objective The goal of this study is always to explore achievable molecular and cellular mechanisms involved in the development of resistance to Herceptin in breast cancer patients.
Background Herceptin can be a humanized monoclonal antibody targeted against the human epidermal development element receptor c erbB two which is overexpressed in roughly 2530% of invasive breast cancer. Herceptin recognizes an epitope on the extracellular domain of c erbB pan p38 MAPK inhibitor two and blocks downstream signaling. Around 50% of individuals respond to Herceptin therapy. having said that, the majority of these will demonstrate illness progression inside 1 year of treatment initiation. Numerous molecular mechanisms contributing to Herceptin resistance have already been proposed. This study aims to define the effects of Herceptin on subcellular c erbB 2 receptor trafficking. We’ve got created a c erbB 2 plasmid fused to Yellow Fluorescent Protein and an epidermal development aspect receptor fused to Green Fluorescent Protein. Each constructs had been sequenced as well as the appropriate sequence obtained. Both constructs have been shown to react with certain antibodies and to possess the predicted molecular weight applying western blotting. Techniques Each EGFR GFP and c erbB 2 YFP plasmids were made use of to transiently transfect COS 7 cells. Time course studies employing low light fluorescent microscopy revealed maximal membrane receptor expression involving 18 and 24 hours following transfection.
Accordingly, we observed 4T1 tumors to exhibit robust staining for the macro phage marker F480, a result that was not recapitulated with PF 562271 administration. As a result, we show for the first time that, as well as the important roles FAK plays in directing carcinoma cell function and behavior, the PTK activ ity of FAK is also clearly necessary for regulating innate immunity within the microenvironments of establishing and progressing mammary tumors. We subsequent applied the 4T1 TR II model to access the distinct part of FAK in TGF driven breast cancer metastasis. As shown in Figure 7a, FAK depletion had no impact on main tumor development of 4T1 TR II cells. Furthermore, despite the fact that FAK defi cient 4T1 TR II cells had been nonetheless very metastatic, FAK deple tion did drastically reduce the immediate pulmonary dissemination of 4T1 TR II cells.
These information sug gest that FAK selectively regulates the initial actions of tumor cell dissemination stimulated by TGF,a outcome that may be consist ent with our findings on the requirement selelck kinase inhibitor of FAK in mediating EMT stimulated by TGF and preventing pri mary colonization of breast cancer cells inside the lung, but not their secondary outgrowth. Moreover, we located no differences inside the MEK162 dissolve solubility potential of manage or FAK deficient 4T1 cells to colonize the lungs following their injection in to the tail vein of BalbC mice. Taken together, these data recommend that the cou pling of TGF to FAK promotes the initial invasion and exit of breast cancer cells from the principal tumor web site. In addition, and similar to handle 4T1 cells, PF 562271 administration commence ning 1 week soon after engraftment of 4T1 TR II cells significantly lowered their growth in mice.
on the other hand, this exact same therapy protocol had no effect around the subsequent metasta sis of 4T1 TR II cells. Collectively, these findings give the initial evidence that FAK activity could be inhibited chemotherapeutically as an efficient two pronged strategy to lessen the growth and metastasis of breast cancers. Furthermore, these benefits also show that amplified TGF signaling in breast cancer cells is capable of driving early tumor cell dissemination from the major mam mary tumor. Discussion TGF is actually a principal player involved in suppressing mammary tumorigenesis by sustaining the composition of standard MEC microenvironments through its ability to inhibit the proliferation and survival of normal MECs. In stark contrast, mam mary tumorigenesis has evolved a variety of mechanisms capa ble of subverting the tumor suppressing functions of TGF and of conferring oncogenic and metastatic properties on this multifunctional cytokine.
In addition to IGF 1 and insulin receptors, mammary epithelial cells may also express insulin IGF 1 hybrid receptors. Hybrid receptors have already been detected in most tissues that express each insulin receptor and IGF 1 receptor. An IGF 1 concentration of two. six nM won’t activate the insulin receptor, but could potentially cause the activation from the insulin IGF 1 hybrid recep tors. Data presented in Figure 3C supports this hypoth esis and suggests that IGF 1 signaling has led to the formation of insulin IGF 1 hybrid receptors. Functional studies with hybrid receptors demonstrate that they behave far more like IGF 1 receptors in lieu of insulin receptors simply because they bind IGF 1 with a a lot greater affinity than insulin. As anticipated, we did not observe activation from the hybrid receptor with ten nM insulin.
Although the significance from the hybrid receptors in mammary epithelial cells in unclear, we hypothesize that the insulin IGF 1 hybrids could be much more abundant in MCF10A cells selleck chemical than otherwise anticipated and this hypothesis is supported by reports that insulin and hybrid insulin IGF 1 receptors are important regulators of breast cancer cells. Throughout this study, we are going to refer to the IGF 1R mediated induction in LIP for simplicity, however the reader really should realize that hybrid receptors could also be involved in regulation of LIP LAP. Due to the fact LIP expression is analyzed 16 hr soon after addi tion of ligand, we also checked p EGFR expression at this later time point. EGFR was not phosphorylated in MCF10A cells or MCF 7 cells 16 hr just after addition of IGF 1 To confirm that IGF 1 was indeed activating the IGF 1R signaling cascade, we analyzed p IGF 1R and p Akt expression at 20 min and 16 hr.
To further assess the possibility that EGFR activity may perhaps play a function inside the IGF 1R stimulated boost in LIP expression, we tested the sensitivity of IGF 1 treated MCF10A cells to the selective EGFR kinase inhibitor, MAPK activation AG1478. Pretreatment of cells for 30 minutes with 0. 1, 1 or five uM AG1478 before addition of 2. six nM IGF 1 for 16 hr didn’t inhibit or decrease the IGF 1 mediated increases in LIP expression and did not inhibit the raise in the LIP LAP ratio. As a manage, five uM AG1478 did lead to the expected reduce in p EGFR, decreases in EGF mediated LIP expression and the LIP LAP ratio, and lesser reductions with 0. 1 and 1 uM. Therapy of cells with 0. 1, and 1.
0 uM AG1478 effectively decreased IGF 1 induced Erk1 two phosphorylation and as expected EGF induced Erk1 two phosphorylation. These data demonstrate that inhibition of EGFR kinase activity reduces IGF 1R mediated Erk1 two activity and suggest that IGF 1R and EGFR signaling crosstalk in MCF10As to regulate Erk1 two activity. Our data also demonstrate that inhibition of EGFR signaling with AG1478 does not inhibit IGF 1R induced Akt activity but does block EGF induced Akt activity.
Conclusions Within this study, we reported here that ET 1 ET receptor program exerts its effects on COX 2 gene expression and PGE2 release in mouse bEnd. 3 cells. The Gi and Gq protein coupled ETB receptor, ERK1 2, p38 MAPK, JNK1 two, and NFB cascades cooperatively mediated these effects of ET 1. These findings regarding ET 1 induced COX 2 PGE2 system imply that ET 1 may possibly play a crucial function in brain in jury, vascular inflammation, and CNS illnesses, mediated by means of MAPK dependent activation of NFB pathway in bEnd. three cells. Pharmacological approaches suggest that tar geting COX 2 PGE2 program and their upstream signaling elements really should yield useful therapeutic targets for brain injury and inflammatory ailments. Strategies Components Dulbeccos modified Eagles medium F 12 medium, fetal bovine serum, and TRIzol were from Invitrogen.
Hybond C membrane and enhanced chemiluminescence Western blot reversible microtubule inhibitor detection method have been from GE Healthcare Biosciences. Anti COX two monoclonal anti physique was from BD Transduction Laboratories. Phospho ERK1 2, phospho p38, phospho JNK1 2 antibody kits were from Cell Signaling. p65, p42, p38, and JNK1 antibodies were from Santa Cruz. Anti glyceraldehyde 3 phosphate dehydrogenase antibody was from Biogenesis. BQ 123, BQ 788, GP antagonist 2, GP antagonist 2A, U0126, SB202190, SP600125, and Bay11 7082 have been from Biomol. Bicinchoninic acid protein assay reagent was from Pierce. ET 1, enzymes, and other chemical substances were from Sigma. Mouse brain microvascular endothelial cell culture Mouse brain microvascular endothelial cells have been purchased from Bioresource Collection and Re search Centre and grew in DMEM F 12 containing 10% FBS and antibiotics at 37 C in a humidified 5% CO2 atmos phere.
The cell line is acquired from mouse BALB c strain brain cerebral cortex endothelial polyoma selleck middle T antigen transformed, which was performed STR PCR profile at BCRC. All the experiments were performed employing this cell line and approved by the ethic approval of Chang Gung University. Confluencent cells have been released with 0. 05% trypsin 0. 53 mM EDTA for 5 min at 37 C. The cell suspension was plated onto 6 well culture plates or ten cm culture dishes for the measurement of pro tein or RNA expression, respectively. Culture medium was changed right after 24 h and then each and every three days. Experi ments had been performed with cells from passages 5 to 13.
Preparation of cell extracts and Western blot analysis Development arrested cells had been incubated with ET 1 at 37 C for several time intervals. The cells were washed with ice cold phosphate buffered saline, scraped, and collected by centrifugation at 45,000 ? g for 1 h at 4 C to yield the entire cell extract, as previously described. Samples were analyzed by Western blot, transferred to nitrocellulose membrane, and then incubated over evening making use of an anti COX 2, phospho ERK1 2, phospho p38 MAPK, phospho JNK1 two, p42, p38, JNK1, p65, or GAPDH antibody.
This method was essential since the pellets didn’t include conveniently detectable levels of N WASP. As previously described, the SH3 domain of cortactin was able to pull down N WASP in WT cells but not N WASP deficient cells. This argues in favor in the conclusion that the N terminal area of cortactin is involved in binding Tir, when the SH3 domain is involved in binding N WASP. Discussion Cortactin is really a scaffold protein implicated in a lot of cellular processes considering that it straight contributes to cytoskeleton remodeling. Cortactin also has oncogenic properties as a consequence of its part in controlling invadopodia formation and cell migration. Additionally, cortactin has emerged as an impor tant target of many pathogens, which includes enteropath ogenic E. coli that manipulate the actin cytoskeleton as a way to invade the host and propagate there.
EPEC cause extreme diarrheal illness in humans by colonizing the gut mucosa and creating A E lesions. EPEC attach to mammalian intestinal cells and induce reorganization with the actin cytoskeleton into pedestal natural compound library like structures beneath neath the bacteria. A crucial event for pedestal formation could be the insertion into the host cell membranes on the EPEC effector Tir, that is initially injected into the cell by a type III secretion method. Tir mimics signaling pathways of your infected cell. Therefore it might serve as a effective model sys tem to study eukaryotic transmembrane signaling. In truth, the Tir Nck N WASP pathway will be the principal one particular by way of which actin polymerizes in EPEC pedestals. These reasons prompted us to study cortactin signaling through EPEC infection making use of N WASP deficient cells.
Although cortactin localizes to pedestals and its truncated forms exert a dominant unfavorable effect, its function just isn’t clear. One example is, does cortactin on its own contribute to actin polymerization in pedestals Our transfection exper iments with selleckchem the GFP W22A cortactin point mutant dem onstrate that cortactin binding and activation from the Arp2 3 complex is vital for pedestal formation, which sug gests that cortactin certainly contributes to efficient actin polymerization. A complementary study employed a similar approach to examine the function of cortactin domains on pedestal formation. It reported identical final results to ours concerning WT cortactin and the mutant W525K. However, the W22A mutant was not studied in that work. To address the part of Erk and Src phosphorylation of cort actin, we used each phosphorylation mimicking and non phosphorylatable mutants, preceding studies have employed only the former. Hence, we have been able to detect a neutral impact on pedestal formation of mutant that mimics phosphorylation by Erk, while the Erk non phosphorylatable kind blocked pedestal formation.