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. Author manuscript; available in PMC: 2012 Sep 7.
Published in final edited form as: Cell Metab. 2011 Sep 7;14(3):378–389. doi: 10.1016/j.cmet.2011.06.015

Figure 3. Knockdown of CDP138 in live 3T3-L1 adipocytes inhibits insulin-stimulated membrane fusion between GLUT4 storage vesicles (GSV) and the PM, but not GLUT4-EGFP trafficking to the TIRF zone.

Figure 3

(A): Schematic illustration of the molecular probes and TIRF microcopy-based live cell assays for GLUT4 trafficking and GSV - PM fusion. (B) and (C): The effect of CDP138 knockdown on insulin-stimulated IRAP-pHluorin insertion into the PM (B), and accumulation of GLUT4-EGFP in the TIRF zone (C). Adipocytes (day 4) were transfected by electroporation with plasmid DNA encoding IRAP-pHluorin or GLUT4-EGFP, together with either the scrambled siRNA or smartPool siRNA against mouse 5730419I09Rik. Cells were reseeded for 72 hrs, serum starved for 2 hr then stimulated with 100 nM insulin for 30 min. Analyses were performed in a cell warmer adapted for a Nikon TiE with fully motorized combined dual laser (488 and 561nm). Images were acquired every 3 min immediately after addition of insulin and analyzed as described in the S.I.. Perfect focus system and multiple points capture program were used to acquire images from multiple positively transfected cells at each time point. Data are mean ± SEM of three independent experiments (n=3) with total 159 cells (43,46,70/exp, Scr siRNA) or 154 cells (43, 37, 74/exp, CDP138 siRNA) in the GLUT4-EGFP trafficking assay; and 125 cells (41, 48, 36/exp, Scr siRNA) or 120 cells (43, 46, 31/exp, CDP138 siRNA) in the GSV - PM fusion assay. P value: CDP138 siRNAs vs scrambled siRNA. Live cell movies for IRAP-pHluorin membrane fusion assay are provided in S.I. See also Figure S3, Movie S1 and Movie S2.