Figure 2: Gβ2 physically interacts with Mfn1 to induce mitochondrial aggregation.

(a) Immunoprecipitation (IP) of Gβ2 with Mfn1. Lysates either from HeLa cells, or cells expressing a shRNA specific to Gβ2 or the latter reintroduced with a mutant of Flag-Gβ2 carrying a mutation for eliminating the RNAi target sequence (the right two lanes marked as asterisk), were incubated with a Gβ2 antibody overnight, followed by western blot with an anti-Mfn1 antibody. The crude lysates from these cells were used as input. (b) Mfn1 coimmunoprecipitates with Gβ2. The cell lysates were incubated with Mfn1 antibody overnight, followed by western blot with an anti-Gβ2 antibody. Mfn1, Gβ2 and heavy chain (HC) levels were quantified by densitomery. Relative intensity were obtained as intensity of bands of Mfn1 (opened box) or Gβ2 (filled box) divided by intensity of heavy chain of relative lines. Error bars represent the s.e.m., n=3; *** for P<0.0005, two-tailed test. (c) Images of fluorescence intensity from the cells co-transfected with CFP-Mfn1 and YFP-Gβ2 in CFP and YFP channels before and after photobleaching of YFP. The increase of the CFP fluorescence and dramatic decrease of the YFP fluorescence were observed after the photobleaching, Scale bar, 10 μm. (d) The histogram shows the FRET efficiency from CFP to YFP in the cells transfected with both CFP-Mfn1 and YFP-Gβ2, CFP-Mfn1 and YFP-Gβ1, CFP–YFP fusion protein and both of CFP and YFP, respectively. Error bars represent the s.e.m., n=18; Differences in the mean FRET efficiency between CFP-Mfn1 and YFP-Gβ2, CFP-Mfn1 and YFP-Gβ1, or CFP and YFP were statistically significant at ***P<0.0005, **P<0.005, two-tailed t-test.