FIG. 4.
DHHC3 and -7 are necessary for Gαq/11 palmitoylation and PM localization. (A) DHHC3 and -7 siRNAs downregulate targeted mRNA expressions. HeLa cells were transfected with a control or DHHC3 or -7 siRNA duplex. At 72 h after transfection, the expression of DHHC3 or -7 mRNA was quantitated by real-time RT-PCR. The expression of DHHC3 and -7 was normalized to that of GAPDH. Error bars show ±SD (n = 3). (B) HEK293T cells were transfected with the indicated siRNAs, and cells were labeled with [3H]palmitate for 4 h. Endogenous Gαq/11 was then immunoprecipitated, followed by fluorography and Western blotting. (C) HeLa cells were transfected with the indicated siRNAs and doubly stained with anti-Gαq/11 (green) and anti-β-catenin (red) antibodies. Note that knockdown of DHHC3 and/or DHHC7 specifically impairs PM localization of Gαq/11. Graphs indicate fluorescent intensity along white lines. Arrows mark the cell-to-cell contact sites along the white line. Scale bar, 20 μm. (D) siRNAs to human-specific DHHC3 and DHHC7 were cotransfected into HeLa cells together with wild-type (WT) GFP-mDHHC3 or catalytically inactive GFP-mDHHC3(C157S) [mDHHC3(CS)]. Cells were immunostained with anti-GFP (blue), anti-Gαq/11 (green), and anti-β-catenin (red) antibodies. The colors are pseudocolors. Scale bar, 20 μm. Relative fluorescence intensities of PM to cytosol are shown (right graph). Error bars show ±SD (n = 20). **, P < 0.01. IB, immunoblotting.