Fig 1: Detection of the interaction between Arf1 and the AP-1 subunit ß1 in HEK293T cells using BRET. (A) HEK293T cells were transfected with a fixed concentration of Arf1-RlucII (black curve), Arf1Q71L-RlucII (red curve), Arf1T48S-RlucII (blue curve) or Arf1T48S,Q71L-RlucII (green curve) and increasing concentrations of ß1-GFP10. Fluorescence and BRET2 were measured in parallel. BRETnet levels are plotted as a function of the ratio of fluorescence over luminescence (GFP10/RlucII). Data points are mean ± SEM of triplicates. (B) BRET50 for each Arf1 construct are shown as mean ± SEM of 4 independent experiments analyzed by ANOVA followed by Tukey’s post-hoc test. *P < 0.05; ***P < 0.001; ns, not significant. (C) Arf1Q71L-RlucII was co-transfected with increasing quantities of full-length ß1-GFP10 (black curve) or truncated ß11–584-GFP10 (blue curve) and BRET2 signals were measured. Data points are mean ± SEM of triplicates. (D) BRETmax and (E) BRET50 are represented as the mean ± SEM of three independent experiments analyzed by paired student t-test to assess statistical significance. *P < 0.05; ns, not significant.
Fig 2: The back-side region of Arf1 participates in the allosteric activation of AP-1 in vivo. (A) HEK293T cells transfected with ?-RlucII/µ1-GFP10 or ß1-RlucII/ß1-GFP10 were treated with vehicle (grey bars) or Brefeldin A (BFA, black bars) and BRET signals were measured. Data are represented as the mean ± SEM of three independent experiments analyzed by paired student t-test to assess statistical significance. ***P < 0.001; ns, not significant.(B) ?-RlucII and µ1-GFP10 were transfected in HEK293T cells with or without Arf1Q71L-HA or Arf1Q71L,W172D-HA and BRET signals were measured. BRET signals shown are observed at similar levels of fluorescence/luminescence ratios. Data are shown as the mean ± SEM of six independent experiments analyzed by one-way ANOVA followed by Tukey’s post-hoc test to assess statistical significance. **P < 0.01; ***P < 0.001; ns, not significant. (C) The level of expression of Arf1Q71L-HA and Arf1Q71L,W172D-HA were determined by Western blot using an anti-HA antibody.
Fig 3: The back-side region of Arf1 does not contribute to AP-1 binding in vivo. (A) BRET titration curves were generated by transfecting HEK293T cells with Arf1Q71L-RlucII (black curve), Arf1Q71L,W172D-RlucII (blue curve) or Arf1Q71L,A136,137H-RlucII and increasing quantities of ß1-GFP10 and BRET signals were measured. (B) BRET50 for each Arf1 construct are shown as the mean ± SEM of 3 independent experiments analyzed by ANOVA followed by Tukey’s post-hoc test. ns, not significant.
Fig 4: Functionality of the RlucII and GFP10 constructs used for BRET. (A) Arf1 recruitment to membranes was measured in HEK293T cells transfected with either Arf1-RlucII or Arf1Q71L-RlucII. Cytosol (grey bars) and membrane compartments (black bars) were separated by cell fractionation and the luminescence was measured in each fraction. Results are expressed as a % of total luminescence. (B) Co-immunoprecipitation of ß1-GFP10 with endogenous ? was detected in HEK293T cells transfected with ß1-GFP10. ß1-GFP10 was immunoprecipitated with an antibody against GFP and endogenous ? was detected by Western blot (Wb) using an anti-? antibody. Western blot images were cropped for space considerations.
Fig 5: Reduced interaction between AP-1 and Arf1 mutants detected by BRET. Arf1Q71L-RlucII or various switch 1 (A) or switch 2 (C) mutated forms of Arf1 were transfected at a low and constant amount along with increasing quantities of ß1-GFP10 into HEK293T cells. The BRET2 signals were measured. BRET50 for each switch 1 (B) or switch 2 (D) mutated forms of Arf11 are shown as the mean ± SEM of 5 independent experiments analyzed by ANOVA followed by Dunnett’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
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