Fig. S1.

(Related to Fig. 1) TRPM7 is in acidic intracellular vesicles. (A) Confirmation of genetic tagging of endogenous TRPM7 with GFP. Reciprocal anti-GFP (Left) and anti-TRPM7 (Right) immunoprecipitations (IP) of TRPM7 from ES clones expressing endogenous WT (BO5 parental ES cell line) or GFP-TRPM7 (clones 29 and 33). Immunoprecipitates were analyzed by Western blotting for GFP or TRPM7. (B and C) HEK293 cells expressing TRPM7 tagged with pHluorin (placed in the S1/2 loop) were fixed and immunolabeled with anti-GFP and 5 mM gold-conjugated protein A. Quantification of immunogold labeling in untransfected control cells or cells expressing pHluorin-TRPM7 (vesicular vs. mitochondrial) (B) and M7V size distributions (C). Diameters of vesicular structures were quantified from 35 images. (D) HEK293 cells expressing pHluorin-TRPM7 were treated with 50 mM NH₄Cl (pH 7.0) to alkalinize intracellular compartments, resulting in an increase in vesicular pHluorin fluorescence.