Rab10 and Rab14 mediate GLUT4 translocation in parallel. 3T3-L1 cells infected with HA-GLUT4-EGFP and scrambled shRNA or Rab10 shRNA were differentiated and then transfected with or without Rab14 siRNA. 48 h after transfection, insulin-stimulated GLUT4 translocation was measured using TIRF microscope. (A) Western blots showing knockdown efficiency of Rab10 and Rab14. (B) Loss of Rab10 and/or Rab14 does not change GLUT4 distribution under basal conditions. GLUT4 distribution under basal conditions was measured by the TIRF/epifluorescence (EPI) ratio, and the ratio was normalized to the control value. (C) The effects of loss of Rab10 and/or Rab14 on GLUT4 translocation. Insulin-stimulated GLUT4 translocation was indicated by TIRF image intensities (I) at different time points normalized to the intensity measured before insulin perfusion (I_{0 min}). (D) Restoring Rab10 or Rab14 recovered part of GLUT4 translocation. TagRFP-Rab10 or TagRFP-Rab14 (both of human origin) was cotransfected with Rab14 siRNA into differentiated adipocytes, and GLUT4 translocation was measured 48 h after transfection. Data are represented as mean ± SEM (error bars). Scrambled, n = 37 cells; shRab10, n = 44 cells; siRab14, n = 31 cells; shRab10+siRab14, n = 33 cells; hRab10, n = 26 cells; hRab14, n = 29 cells.