Fig. 7
From: The a subunit isoforms of V-ATPase are involved in glucose-dependent trafficking of insulin granules
Localization of insulin granules in control and a3KD MIN6 cells. (A) Expression of a3 in a3KD cells. Cells were transduced with pSIREN-RetroQ-negative control shRNA (control) or pSIREN-RetroQ-a3-shRNA (a3KD). a3KD cells were further transduced with pMX-V5-a3 (a3KD + V5-a3). Endogenous a3 and V5-a3 were detected with an antibody specific for a3 (left upper panel). β-actin was also detected using a corresponding antibody (left lower panel). Original blots are presented in Supplementary Fig. 12. Relative signal intensities of a3 were calculated as means ± S.E. from three independent experiments compared with control cells (set to 100%) (right panel). (B) Localizations of insulin and V5-Rab27A in control (upper panels) and a3KD (lower panels) cells. Cells expressing V5-Rab27A were sequentially incubated with 2.2 mM glucose for 60 min, 20 mM glucose for 5 min, and 2.2 mM glucose for 5 min, and then stained with antibodies specific for insulin (magenta) and V5 (green). Merged images are also shown. (C–E) Cells with peripheral insulin and Rab27A according to the glucose concentration. The percentage of cells with peripheral insulin (C, E) or Rab27A (D) was calculated as described in Fig. 2B. Control cells, open squares (insulin), open circles (Rab27A); a3KD cells, closed squares (insulin), closed circles (Rab27A); a3KD#1 + V5-a3 cells, shaded squares (insulin). Data are means ± S.E. from three independent experiments. n > 30 cells in each experiment. *p < 0.05, (A) unpaired two-tailed Student’s t-test, (C–E) Unpaired multiple t test (Holm-Šídák method).
