Fig. 3

Localization of insulin granules in a2KD MIN6 cells. (A) Expression of a2 in a2KD#1 cells. MIN6 cells were transduced with pSIREN-RetroQ-negative control shRNA (control) or pSIREN-RetroQ-a2-shRNA#1 (a2KD#1). a2KD#1 cells were further transduced with pMX-V5-a2 (a2KD#1 + V5-a2). Endogenous a2 and V5-a2 were detected with an antibody specific for a2 (left upper panel). β-actin was also detected using a corresponding antibody (left lower panel). Original blots are presented in Supplementary Fig. 10. The blots were cut horizontally at ~ 75 kDa. Upper and lower membranes were subjected to hybridization with anti-a2 and anti-β-actin antibodies, respectively. A full-length membrane was cut to conserve the anti-a2 antibody, which is commercially unavailable. Relative signal intensities of a2 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 a2KD#1 (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); a2KD#1 cells, closed squares (insulin), closed circles (Rab27A); a2KD#1 + V5-a2 cells, shaded squares (insulin). Data are means ± S.E. from more than 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).