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. 2013 Jun 17;110(27):11011–11016. doi: 10.1073/pnas.1309531110

Fig. 1.

Fig. 1.

Localization of TRPC3 in mitochondria. (A) TRPC3 was found in the homogenate (h), mitochondrial (m), or cytosolic (c) fractions isolated from rat liver (Left) and brain (Right). HSP60, VDAC protein, and cytochrome (cyto) C served as mitochondrial markers, calnexin as an ER marker and β-actin as a loading control. Polyclonal anti-TRPC antibodies were used. (B) TRPC3 immunoreactivity detected by the monoclonal anti-TRPC3 antibody in homogenates (h), cytosolic (c), and mitochondrial (m) fractions of the cerebellum from the WT but not Trpc3−/− (KO) mice. HSP60, VDAC, and cyto C served as mitochondrial markers, calnexin as an ER marker, EEA1 as an endosome marker, Lamp1 as a lysosome marker, TrR as plasma membrane marker, and β-actin as a loading control. For Western blotting, all isolated fractions from WT and Trpc3−/− tissues were run on the same gel and blotted on the same membrane. (C) Immunocytochemical staining of cerebella from WT and Trpc3−/− mice using the polyclonal anti-TRPC3 antibody (green; Alomone. Cat. No. ACC016, Lot No. AN0702) and monoclonal anti-MAP2 antibody (red). Hochest33258 was used to label nuclei (blue). (Scale bars, 20 µm.) (D) Immunofluorescent labeling of TRPC3 in mitochondria. Representative images of HeLa cells stained with the polyclonal anti-TRPC3 antibody (green; from C. Montell) and loaded with 10 nM MitoTraker red (red). (Scale bars, 5 µm.) 40×, 40× objective; 40× 3×, 40× objective with 3× zoom. (Right) Summary of percent fluorescence intensity of actin, TRPC3, or HSP60 colocalized with MitoTraker red over the total fluorescence intensity of the corresponding protein, n = 10 for actin and TRPC3, respectively, n = 3 for HSP60. Representative images for actin and HSP60 are shown in Fig. S2A. (E) (Left) Immunobloting of TRPC3 in the intact mitochondria and mitoplast of rat liver cells. (Right) Immunobloting of TRPC3 in the swelled mitochondria incubated with or without proteinase K in 0.5% TritonX-100 or not. Bcl-2 served as an outer membrane marker, cyto C a mitochondrial intermembrane space marker, PRODH a mitochondrial inner membrane marker, and Hsp60 a matrix marker. Cytosolic (c), mitochondrial (m), mitoplasts (mp), and proteinase K (Prot. K) (F) Electron microscopic images (Left) of immunogold labeling by the polyclonal anti-TRPC3 antibody (Alomone Cat. No. ACC016, Lot No. AN0702) of mitochondrial inner membranes (red arrows) of the cerebellum from WT, but not Trpc3−/− (KO), mice. Labeling to other membrane structures, presumably plasma membrane, is indicated (red arrowhead). (Scale bar, 200 nm.) Statistics of gold particles associated with mitochondrial inner membranes per field (Center) and that associated with all membrane structures per field (Right) are shown as means ± SEM of 50 EM fields from three mice for each genotype.