Effect of p38 MAPK on SAC1 oligomerization and constitutive secretion. (A) Expression of constitutively active alleles of MKK3 and 6 induces ER localization of SAC1 in serum-starved cells. COS7 cells were transfected with plasmids for expressing GFP-SAC1 (green) and flag-MKK6(Glu) (blue) or flag-MKK3(Glu) (blue). Cells were fixed, permeabilized, and costained with M2 antibodies (blue) and polyconal anti-TGN46 (red). Merged images show colocalization of GFP-SAC1 (green) and TGN46 (red). Bar, 15 μm. (B) COS7 cells transiently expressing flag-SAC1 and GFP-SAC1 were starved in the presence of 0.5% serum for 24 h and then stimulated with 10% serum with or without 10 μM SB203580. Cells were lysed and flag-SAC1 was immunoprecipitated with M2 agarose beads. Proteins were separated by SDS-PAGE followed by immunoblotting with anti-GFP or M2 antibodies. (C) COS7 cells transiently expressing flag-SAC1 and GFP-SAC1 were starved in the presence of 0.5% serum for 24 h and then stimulated with 10% serum in the presence of increasing concentrations of SB503280 (0.3–3 μM). Cells were lysed and flag-SAC1 was immunoprecipitated with M2 agarose beads. Proteins were separated by SDS-PAGE followed by immunoblotting with anti-GFP or M2 antibodies. GFP-SAC1 bands were quantified and compared with the relative levels of precipitated GFP-SAC1 from starved nonstimulated cells. Data represent means ± SD from three independent experiments. (D) NIH3T3 cells were grown as indicated. Pulse-chase analysis of protein secretion was conducted as described in Materials and methods. Data represent means ± SD from three independent experiments. (E) COS7 cells were transfected with a control plasmid without insert or with a plasmid for expressing flag-MKK3(Glu). Cells were serum-starved for 24 h and pulse-chase analysis of protein secretion was conducted as described in Materials and methods. Data represent means ± SD from three independent experiments.