Fig. 5
Assessment of fractional binding using single-molecule approach. a Diagram showing experimental workflow. b Cleared cell lysates from the indicated double knock-in cells were spread between quartz slides and glass coverslips for TIRF imaging. The GFP and Halo + JF585 fluorophores were excited simultaneously with 488-nm and 561-nm lasers at 12.5 Hz for 40 s. Scale bar = 1 µm. c Number of photobleaching steps detected for SEC5-Halo and SEC8-GFP in experiments shown in panel b. d Quantification of the fraction of subunit A bound to (–> ) subunit B from experiments shown in panel b. Numbers of particles analyzed for each pair are shown in Supplementary Fig. 4c. e Fraction of SEC5-Halo and SEC8-GFP bound to each other in SEC3 or SEC6 shRNA-treated cells compared to control. Center line denotes mean. f Western blot analysis of SEC3 and SEC6 shRNA knockdown efficiencies in the experiment shown in e. Error bars denote  ± SD. Statistical significance was computed by one-way (d) or two-way (e) ANOVA, followed by Tukey’s multiple comparison tests. Experiments were repeated three times and data were pooled
