Mutation of the Rab1-binding site in OPTN does not affect its dimerization or binding to LC3 or ubiquitin. (A) Micrographs of beads coated with GST-4×ubiquitin (GST-4xUb) and incubated with either 1 μM GFP-OPTN (WT) or versions with mutations in the Rab1 or ubiquitin-binding sites. Each large datapoint in the bar graph depicts the average mean fluorescence intensity on a selection of beads and represents an independent experiment (n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (B) Micrographs of beads coated with GST-LC3B and incubated with either 1 μM GFP-OPTN (WT) or versions with mutations in the Rab1 or LC3 (LIR)-binding sites. Each large datapoint in the bar graph depicts the average mean fluorescence intensity on a selection of beads and represents an independent experiment (n = 3), with smaller gray datapoints representing all the technical replicates (AU, arbitrary units). The mean ± SD is indicated. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). (C) Immunoblot of cell lysates of pentaKO HeLa cells stably expressing WT OPTN or the OPTN Rab1-binding mutant (representative of three repeats). Lysates were incubated with increasing concentrations of the cross-linker bis(sulfosuccinimidyl)suberate (BS3) at RT and quenched at 50 mM Tris-HCl prior to SDS-PAGE and blotting. With cross-linker, OPTN dimers are readily detected. GAPDH is a loading control and a negative control as it does not readily dimerize. Source data are available for this figure: SourceData FS3.