FIG. 2.

Effect of insulin on translocation of TRPV2 in MIN6 cells. A: Effect of exogenous insulin on translocation. MIN6 cells transfected with TRPV2-GFP were preincubated with KRB buffer containing 2.7 mmol/l glucose for 1 h. They were then incubated with 10 nmol/l insulin for 30 min, and fluorescence images before (a) and after stimulation (b) are shown. B: Localization of TRPV2. a: Colocalization of TRPV2 with an ER marker. Fluorescence images were obtained in unstimulated MIN6 cells expressing TRPV2-RFP (red) and ER-YFP (green). Under a basal condition, most of the TRPV2 signals colocalized with the ER marker. b and c: Colocalization of TRPV2 with a plasma membrane marker. Fluorescence images were obtained in unstimulated (b) and insulin-stimulated (c) MIN6 cells expressing TRPV2-RFP (red) and PM-YFP (green). Under a basal condition, the TRPV2 signal was not colocalized with the PM marker, whereas some of the TRPV2 signals were colocalized with the PM marker in insulin-stimulated cells. C: Effect of insulin on translocation of c-myc–tagged TRPV2. MIN6 cells transfected with c-myc–tagged TRPV2 were preincubated with KRB buffer containing 2.7 mmol/l glucose for 1 h. They were then incubated for 30 min with (b) or without (a) 10 nmol/l insulin. The c-myc epitope was stained in intact cells. Bar: 20 μm. D: Quantification of the immunoreactivity of c-myc epitope. MIN6 cells transfected with c-myc–tagged TRPV2 were incubated for 30 min in various conditions, and cell surface expression of c-myc was quantified. Anti-Ins, anti-insulin antibody. *P < 0.05 vs. none; **P < 0.01 vs. none. E: Effect of BAPTA loading on basal secretion of insulin. MIN6 cells loaded with or without BAPTA were incubated for 60 min in the presence of 2.7 mmol/l glucose, and insulin secretion was measured. Values are the mean ± SE for four experiments. *P < 0.05 vs. control. F: Effect of serum on translocation of c-myc–tagged TRPV2 in CHO cells. CHO cells transfected with c-myc–tagged TRPV2 were incubated for 30 min with (b) or without (a) 10% serum. The c-myc epitope was stained in intact cells. Bar: 20 μm. (Please see http://dx.doi.org/10.2337/db08-0862 for a high-quality digital representation of this figure.)