Fig. 5.

PI4K2A is required for VAMP3 interaction with cognate Q-SNARE Vti1a. (A) COS-7 cells were transfected with control siRNA or siRNA directed against PI4K2A for 2 days, fixed, then permeabilized and incubated with a rabbit polyclonal antibody directed against PI4K2A and a mouse monoclonal antibody directed against Vti1a. (B) The cartoon depicts the proposed role of PI4K2A (blue) in sorting of VAMP3 (lavender) to endosomal membranes, where it engages in SNARE complex formation with Vti1a (green) and other cognate Q-SNAREs. NEM inhibits disassembly of the complex, trapping Vti1a bound to VAMP3. (C) COS-7 cells were treated for 2 days with control or PI4K2A siRNA, transfected with GFP–VAMP3, and either preincubated with 1 mM NEM for 30 min or directly lysed. GFP–VAMP3 was immunoprecipitated from total cell lysates using anti-GFP beads. Immunoprecipitated samples were eluted and analyzed by SDS–PAGE, followed by western blotting. (D) Quantification of Vti1a present in complex with VAMP3 in immunoprecipitated samples, as shown in C. Results are mean±s.e.m. from five experiments. *P<0.05 between NEM-treated samples, as determined by one-way ANOVA analysis; N.S. not significant compared with control. (E) Assessment of PI4K2A knockdown from the experiment shown in C. After 72 h of treatment with control or PI4K2A siRNA, cells were lysed and analyzed by western blotting using antibodies against PI4K2A and actin. A.U., arbitrary units. Scale bars: 10 µm.