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. 2014 Sep 1;127(17):3745–3756. doi: 10.1242/jcs.148809

Fig. 3.

Fig. 3.

PI4K2A regulates VAMP3 sorting. (A) COS-7 cells were treated with control siRNA or siRNA directed against PI4K2A for 2 days prior to transfection with VAMP3–HA. At 1 day after transfection, cells were fixed, permeabilized and incubated with rabbit polyclonal antibody against PI4K2A and mouse monoclonal antibody against the HA epitope. PI4K2A knockdown resulted in an accumulation of VAMP3 on enlarged vesicles. (B–D) Live-cell confocal microscopy of COS-7 cells after a 2-day treatment with control or PI4K2A siRNA duplexes and co-transfection with GFP–VAMP3 and either LAMP-1–mRFP (B,C) or mCherry–Rab5, mCherry–Rab7 or mCherry–Rab11 (D). (C) Comparison of GFP–VAMP3 and LAMP-1–mRFP pixel (Pearson's) correlation coefficients. (D) PI4K2A depletion results in missorting of VAMP3 into enlarged LAMP1- and Rab7-containing compartments (control siRNA, n = 27; PI4K2A siRNA, n = 31). Enlargements of the boxed area are shown in the insets for A and B. The boxes in D represent VAMP3-enriched vesicles; arrows indicate Rab colocalization. Scale bars: 10 µm.