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. 2016 Oct 17;291(49):25678–25691. doi: 10.1074/jbc.M116.756544

FIGURE 6.

FIGURE 6.

Effects of 2BP and mutation C38A on the plasma membrane anchorage of TRPP3NT and palmitoylation of TRPP3 at Cys-38. A, representative confocal images showing subcellular localization of EGFP-TRPP3NT (EGFP fused the TRPP3 N terminus, Met-1–Leu-95), EGFP-TRPP3CT (EGFP fused the TRPP3 C-terminal fragment Glu-706–Ser-805, as a negative control), and EGFP-N-Ras (EGFP fused with human N-Ras, as a positive control) in HEK cells. Scale bar, 10 μm. B, subcellular localization of EGFP-TRPP3NT and EGFP-N-Ras in HEK cells without (Ctrl) or with fatty acid starvation, with 100 μm 2BP treatment overnight, or with the C38A mutation. Scale bar, 10 μm. C, palmitoylation of FLAG-TRPP3 in HEK cells detected by immunoprecipitation (IP) acyl-biotin exchange assays. See under “Experimental Procedures” for method details. Cells were transfected with WT FLAG-tagged TRPP3 and mutant C38A or T39E. 2BP treatment was as in B. Palmitoylated TRPP3 proteins were detected with streptavidin-HRP antibody, and total immunoprecipitated TRPP3 proteins (Input) were detected with FLAG antibody. Shown are representative immunoblots (IB) from three independent experiments. D, representative confocal images showing subcellular localization of EGFP-TRPP2NT (EGFP fused the TRPP2 N terminus, Met-1–Lys-215) and EGFP-TRPP2CT (EGFP fused theTRPP2 C terminus, Asp-682–Val-968) in HEK cells. Scale bar, 10 μm.