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. 2020 Mar 5;8:e8751. doi: 10.7717/peerj.8751

Figure 4. The effect of knockdown of VAMP isoforms on HA–GLUT4–GFP translocation.

Figure 4

HeLa cells expressing HA–GLUT4–GFP were grown and transfected with 200 nM scrambled control sequence (SCR), VAMP2, 4 or 8 SMARTpool siRNA as described. Cells were serum-starved (basal) before being treated with or without 1 µM insulin (insulin) for 20 min. Cells were fixed and surface HA was stained as described. (A) Immunofluorescence images of a typical field for each condition are shown. (B) The fold-change in the HA/GFP ratio with insulin-stimulation. Values were compared using Student’s t-test (*p < 0.05 and **p > 0.05) and are means ± SD of 16 random fields of view, taken from four independent experiments. In this analysis, the HA/GFP ratio in the absence of insulin is set equal to 1 for each siRNA. VAMP2 knockdown significantly inhibited insulin-simulated HA–GLUT4–GFP translocation, *p = 0.01. The apparent reduction in translocation upon VAMP4 knockdown did not reach statistical significance. (C) Quantifies the basal HA/GFP ratio for each VAMP knockdown compared to that observed in SCR siRNA treated cells; values represent the means ± SD of 16 fields of view, taken from four independent experiments **p < 0.05.