Fig. 3.

Verification of antibodies specificity. a TBC1D1 and TBC1D4 were immunoprecipitated from a human vastus lateralis muscle sample and the immunoprecipitates (IP) (150 μg) loaded together with the pre-IPs (lysate cleared with beads; 30 μg) and the post-IPs (supernatant fraction; 30 μg). Following SDS-PAGE proteins were transferred to PVDF membranes, which were then probed with TBC1D4 antibody. b Same samples as above (a), but the membranes were incubated with TBC1D1 antibody. c Membranes (a) were re-incubated with a mixture of TBC1D1 and TBC1D4 antibodies and developed to show the separation between TBC1D1 and TBC1D4. d Western blot analyses were performed using an insulin-stimulated human vastus lateralis muscle sample loaded in three wells. The membrane was cut through the middle lane. The two membranes were incubated with different antibodies as indicated. The different pieces of membrane were put back together and developed. TBC1D1 and TBC1D4 ran at different molecular masses, whereas the signal from the PAS antibody aligned with TBC1D4, but not with TBC1D1. e TBC1D4 was immunoprecipitated from the same sample used above (d), and pre-IP (60 μg), post-IP (60 μg) and IP (120 μg) were subjected to SDS-PAGE and transferred to PVDF membranes. These were then incubated with antibodies as indicated. All bands in the IP lane ran with the same, or slightly higher, molecular mass as the pre-IP sample. IB, immunoblot