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Fig. 3. TRPC1 colocalises and co-immunoprecipitates with caveolin-3. (A-H) Skeletal-muscle cryosections (TA) from mdx and WT mice were double labelled for TRPC1 (red) and caveolin-3 (green) (A-F). Images were then overlaid (C,F) and the colocalisation was quantified. (D-F) Higher-magnification images of the insets shown in A-C. Scale bars: A, 50 µm; D, 5 µm. (G) White pixels represent true colocalisation and were compared to the binary images generated from each labelling (data not shown; see Materials and Methods). Staining of caveolin and TRPC1 showed substantial but not complete overlap. (H) More than 50% of TRPC1-stained pixels colocalised with caveolin-3 staining in the sarcolemma and subsarcolemmal compartment, and vice-versa, in both mdx and WT groups. In addition, a subset of TRPC1 staining appeared to be associated with punctate subsarcolemmal structures that stained weakly with anti-caveolin3. Mean ± s.e.m. of ten membrane segments per mouse is represented, from three mice per group. (I-K) Co-immunoprecipitation of TRPC1 and caveolin-3. Total lysate from mdx and WT skeletal muscle was pre-cleared with agarose beads and incubated with anti-TRPC1. Immunocomplex was resolved by a SDS-PAGE and membranes were probed for caveolin-3. (I,J) Caveolin-3 co-immunoprecipitates with TRPC1 in both mdx and WT skeletal muscles. (K) A reverse IP also confirmed that caveolin-3 and TRPC1 co-immunoprecipitate. (I) As negative controls, samples were processed without the primary antibody (anti-TRPC1) or without the lysate. Lysate of a WT mouse was used for the IP.
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