03% SDS with

agitation for 1 h at 4 °C. Cells were harvested and 200 μl of cell lysate was transferred to 1.5 ml tubes and heated for 1 h at 56 °C in the presence of 30 μl of 10% BSA. A 1:1 volume of 50% TCA was added to the samples and incubated at 4 °C overnight with agitation. The precipitated protein complex was subjected to centrifugation at 12,000 × g for 15 min at 4 °C. The protein pellet was washed 2 times with ice cold 750 μl acetone. The dried pellet was dissolved with 300 μl 0.5 N NaOH and heated for 1 h at 65 °C. The total amount of [14C]-labeled protein from duplicate samples was determined using a liquid scintillation counter. 2D-PAGE analysis was performed using selleck chemical the 2-D DIGE technology as previously described [22] with some modifications. Myotubes derived from 10 NGT or 10 T2D individuals, were grown on 150 mm dishes washed 2 times with

cold PBS and once with 250 mM sucrose, and harvested in 2 ml of cold 250 mM sucrose. The cell pellet was lysed in 2-D DIGE lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS pH 8.5). The myotube protein extract (50 μg portion) was incubated with 1 μl of diluted Nuclease Mix (GE Healthcare, 80-6501-42, diluted 1:8 in DIGE lysis buffer) for 30 min at RT. Following nuclease treatment, total protein concentration was determined in each sample using BSA as a standard (RC/DC kit, Bio-Rad, #500-0121). Final protein concentration was adjusted to 4.6 μg/μl selleck screening library with DIGE lysis buffer. An internal standard sample was prepared by pooling small volumes from each sample and used in all gels to control for system related result variation and therefore to minimize the gel-to-gel variation effects. A volume corresponding to 50 μg total protein of the nuclease-treated myotube protein extract was labeled with either Cy3 or Cy5 fluorescent dye, as per manufacturer’s instructions (CyDye DIGE Fluor minimal dye, GE Healthcare, RPK0272, RPK0273 & RPK0275). The nuclease-treated internal standard sample was labeled with Cy2 fluorescent dye. Myotubes from T2D and NGT patients were treated with or without insulin and randomly assigned pheromone to Cy3 or Cy5 labeling. However, data from the insulin stimulated

condition are not reported in this study due to further validation and investigation. Samples were further analyzed by single gels, as described below. Due to the possibility of inter individual variation, all samples from the same individual were processed on one gel. One complete Cy3 and one complete Cy5 labeling reaction mix was combined with an equivalent portion of the internal standard reaction mix. The total volume was adjusted to 45 μl with DIGE lysis buffer (pH 8.5). The 45 μl mixture was further diluted by addition of 40 μl of 2× IPG, DTT sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2%, IPG buffer 3–11, and 2% (w/v) DTT). Samples were loaded onto 24 cm 3-11NL IPG strips previously rehydrated in 450 μl DeStreak solution with 0.