5 to 3 ug plasmid DNA or 50nM siRNA using Lipofectamine2000 and Lipofectamine RNAiMAX according to manufacturers instructions. HEK293T unlike cells were transfected using Inhibitors,Modulators,Libraries polyethyleneimine and ex panded in high glucose Inhibitors,Modulators,Libraries DMEM, 48 hours prior to experiment. All experiments requiring BMP2 stimulation were conducted after 6 hours starva tion in DMEM without serum. Cells were grown on un coated cell culture plastic unless stated otherwise. Expression plasmids The plasmids encoding human BMPRII LF HA or mouse BMPRIb HA were described previously. Single point mutations used to generate kinase dead receptors were generated by cyclic mutagenesis PCR as described in. The construct encoding N terminal flag tagged p55�� was generated by cloning the full length open reading frame of mouse p55�� into the TOPO TA vector before ligation via EcoRINotI into pcDNA3.
1 basic. Cloning primers Inhibitors,Modulators,Libraries used in this paper are available upon request. The construct encoding HA tagged p85 was a kind gift from Bart Vanhaesebroeck. The construct encoding GFP tagged PH domain of Akt was a kind gift from Kerstin Danker. All constructs were verified by DNA sequencing. Immunoprecipitation assays Immunoprecipitation of expressed proteins from HEK293T cells was performed using a modified radio immunoprecipitation assay buffer containing 0. 5% sodium dodecyl sulphate and 0. 1% Nonidet P 40. Immu noprecipitation from C2C12 cell extracts was performed using a modified radio immunoprecipitation assay Inhibitors,Modulators,Libraries with 0. 1% sodium dodecyl sulphate and 0. 5% Nonidet P 40. A detailed description of the immunoprecipitation and immunoblotting procedures can be found in Additional file 7.
PIP bead assay was purchased from Echelon Bio science and precipitation was performed according to manufacturers instructions. Mass spectrometry Identification of p55�� binding Inhibitors,Modulators,Libraries to GST BMPRII was per formed as described in. PIP bead binding proteins were identified by matrix assisted laser desorption ionisation time of flight mass spectrometry based peptide mass finger printing as described previously. Scratch wound healing The scratch wound healing assay was performed using cell culture inserts according to the manu facturers instructions on uncoated tissue culture plastic. A detailed description of the procedure can be found in Additional file 7.
The rate of cell migration was mea sured by quantifying the intensity translocation values for three independent biological replicates per condition using a selective mask filter. Boyden chamber assay selleck chemical The assay was performed in a similar manner to with a detailed description of the procedure in Additional file 7. Chemotaxis assays Two dimensional chemotaxis was assayed using the u slide chemotaxis chamber system according to accompanying instructions with the following modifications 1 day prior to seeding, chambers were coated with 0. 5% gelatin solution in humidified atmosphere washed for 1 hour and dried at 37 C.