Fig. 4

Detection and FRET quantitative analysis of oligomerization of TRIM32. (A) Left panel: Co-IP-mediated analysis of TRIM32 self-interaction. HEK293T cells were transfected with Flag-tagged TRIM32, P130S, R394H, D487N, V591M or P619S and their EGFP-tagged counterparts. Immunoprecipitated Flag-tagged proteins were probed for the presence of conjugated EGFP-tagged proteins by western blot. Right panel: Protein grey value analysis performed using ImageJ, and the data are presented as means ± SEMs. (B) The fluores-cence images of representative cells and the corresponding pseudo-color ED and Rc images as well as their histograms. Scale bar = 15 µm (40 × magnification). (C) The corrresponding ED-Rc plot from at least 100 cells. The saturation binding curves were fitted using Origin with the function: ED = EDmax × Rc/(Kd + Rc). (D) Statistical analysis of ED-Vmax. ED-Vmax is the maximum ED corresponding to saturation of donor binding sites by an acceptor. Student’s T-tests were performed to analyse the data of each variant and WT. all data are shown with error bars (± SEMs). “*” on the histogram columns represents the significant differences between each variant and WT; “*” on the horizontal line represents the significant differences of the variants below it. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance