JCS032003 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. TRPC1-CFP binds directly to caveolin-3-YFP (FRET). C2 myoblasts were transfected with TRPC1-CFP and caveolin-3-YFP plasmids. We used a ratio of 1:3 of caveolin-3-YFP to TRPC1-CPF plasmids for the transfections (1 µg/ml and 3 µg/ml, respectively), based on calibration experiments comparing the plasmid concentration with fluorescence expression. Cells were imaged using low laser power. A ROI was selected and bleached using the acceptor (YFP) laser line (514 nm). Image analysis showed an increase in the CFP fluorescence, confirming the direct binding of TRPC and caveolin-3 (upper panel). Cells transfected with either plasmid were submitted to the same bleaching protocol and showed no increase in the CFP fluorescence upon bleaching of the acceptor (middle and lower panels). Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Exogenously expressed TRPC1-CFP and caveolin-3-YFP are recognised by anti-TRPC1 and anti-caveolin-3 antibodies. C2 myoblasts transfected with either TRPC1-CFP or caveolin-3-YFP were immunolabelled using respective antibodies. Anti-TRPC1 antibody recognized both endogenous (arrowheads) and exogenous TRPC1 (arrows). Non-transfected C2 myoblasts showed low expression levels of TRPC1 and diffuse cytoplasmic labelling. Exogenous TRPC1 localized mainly diffusely in the cytoplasm and also in the paranuclear region, compatible with Golgi localisation. Anti-caveolin-3 antibody was also tested and showed a high level of colocalisation with the exogenously expressed caveolin-3-YFP. Scale bar: 10 µm.