Fig. 5. CFP lifetime of TRPC1-CFP decreases when co-transfected with caveolin-3–YFP into C2 myoblasts. (A) C2 myoblasts were transfected with TRPC1-CFP alone or in combination with caveolin-3–YFP. A reduction in the CFP lifetime was detected when both plasmids were co-transfected, compared with cells transfected only with TRPC-CFP [from 2.7±0.03 nanoseconds (ns) to 2.1±0.03 ns; FRET efficiency 20%; see B,C]. Scale bar: 10 µm. (B) Example of a lifetime measurement showing the shift of the graph to the left (shortening of lifetime) in cells co-transfected with both TRPC-CPF and caveolin-3–YFP. (C) Mean of lifetime from cells transfected with TRPC-CFP only or in combination with caveolin-3–YFP. Dots represent individual cells from three independent experiments. (D) When cells co-transfected with TRPC1-CFP and caveolin-3–YFP were incubated with H₂O₂, an increase in the CFP lifetime was detected (from 2.17 to 2.33±0.03 ns) compared with control cells (no H₂O₂). This increase was prevented by incubation of the cells with PP2 prior to H₂O₂ treatment, which shows that Src activation can induce TRPC1 conformation changes or interfere with the binding properties involved in the interaction between TRPC1 and caveolin-3 (see Discussion). Graph represents mean ± s.e.m. of a minimum of six cells per group per experiment, from three independent experiments. ^***P<0.001.