RNAi-mediated depletion of SAC1 increases constitutive secretion and affects PI(4)P distribution in serum-starved cells. (A) COS7 cells were transfected with siRNAs directed against SAC1 or with mutated control siRNAs. Knockdown efficiency was determined by immunoblotting using anti-SAC1 and anti–glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) antibodies. Cells were serum-starved for 24 h before pulse-chase analysis of protein secretion was conducted as described in Materials and methods. Data represent means ± SD from three independent experiments. (B) COS7 cells were transfected with siRNAs directed against SAC1 or with control siRNAs. Knockdown efficiency was determined by immunoblotting using anti-SAC1 and anti–glyceraldehyde-3-phosphate dehydrogenase antibodies. The cells were then serum-starved for 24 h and incubated in the presence or absence of 10 μM SB203580 for 30 min and then stimulated with serum for another 20 min followed by pulse-chase analysis of protein secretion as described in Materials and methods. Data represent means ± SD from three independent experiments. (C) COS7 cells were cotransfected with siRNAs directed against SAC1 and with plasmids for expressing either RNAi-resistant GFP-SAC1 (GFP-SAC1*, green) or an RNAi-resistant phosphatase-dead SAC1 mutant (GFP-SAC1-C/S*, green). Cells were serum starved for 24 h before being subjected to immunofluorescence microscopy using monoclonal anti-PI(4)P antibodies (red). Bar, 15 μm.