Figure 2.
Effect of TRAPγ/SSR3 deficiency on endogenous proinsulin biosynthesis in β-cells. A: INS1E cells were transfected with scrambled oligo or TRAPγ/SSR3-KD (SSR3i). At 72 h posttransfection, cells were lysed and analyzed by reducing SDS-PAGE and immunoblotted with antiproinsulin and anti-SSR3 antibodies. B and C: Quantitation (mean ± SD) of TRAPγ/SSR3 protein and proinsulin protein from three independent experiments (normalized to tubulin). D: At 72 h posttransfection, control (Ctrl) and TRAPγ/SSR3-KD (SSR3i) INS1E cells were pulse labeled with 35S-Met/Cys for the indicated times. Cell lysates (normalized to trichloroacetic acid–precipitable counts) were immunoprecipitated with anti-insulin and analyzed by reducing SDS-PAGE and phosphorimaging. E: Quantitation (mean ± SD) of newly synthesized proinsulin from five independent experiments like that shown in D, with the proinsulin band from Ctrl INS1E cells at 10 min labeling set to 1. *P < 0.05 compared with Ctrl.