Extended Data Fig. 8: Protein interactors of FAM134B/FAM134C-containing oligomers.
From: Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum
a, HEK 293T cells were transfected with a control plasmid (GFP), V1-FAM134B-WT, V2-FAM134B-WT, V1-FAM134C-WT or V2-FAM134C-WT. Cells were also co-transfected with V1-FAM134B-WT and V2-FAM134B-WT or V1-FAM134C-WT and V2-FAM134C-WT. The GFP trap was analysed by western blot. (n = 1 experiment). b, Ubiquitinated lysine residues identified by proteomics analysis in the RHD of immuno-isolated dimeric FAM134B: diGly peptides were significantly enriched (log2 enrichment > 2.0 and –log10 p value > 1.3, one-tailed unpaired Student’s test). n = 3 independent experiments. Schematic of the FAM134B-RHD showing the localisation of ubiquitinated lysine residues. c, Single-sided volcano plot of the quantitative label-free interactome of FAM134C homodimers and d, FAM134B/FAM134C heterodimers depicting identified RHD-containing ER proteins (blue), autophagy-related proteins (green), and components of ubiquitination machinery (red) (log2 enrichment > 2.0 and –log10 p value > 1.3). Data are means ± s.d. of n = 3 independent experiments. e, Heat map comparing the interaction of RHD-containing ER proteins, autophagy-related proteins and the ubiquitination machinery with WT FAM134B homodimers, FAM134C homodimers and FAM134B/FAM134C heterodimers (immuno-isolated using BiCAP). Interaction partners with log2 enrichment > 2.0 and –log10 p value > 1.3 were plotted. n = 3 independent experiments, one-tailed unpaired Student’s test. f, Venn diagram of interactors of FAM134B homodimers, FAM134C homodimers and FAM134B /FAM134C heterodimers. Numbers represent significantly enriched interaction partners (log2 enrichment > 2.0 and –log10 p > 1.3, one-tailed unpaired Student’s test). n = 3 independent experiments. g, Annotation enrichment analysis of the interactome of FAM134B and FAM134C heterodimers. Bars represent significantly enriched gene ontology biological process (GOBP), gene ontology cellular component (GOCC), gene ontology molecular function (GOMF), and domain enrichment (Pfam). h, Confocal fluorescence microscopy analysis of the BiFC signal produced by interactions between V1-FAM134B and V2-FAM134B. Fixed cells expressing V1-FAM134B and V2-FAM134B were stained for LC3B (red) and p62 (blue). Scale bar = 10 µm. i, Pearson’s correlation coefficients obtained from the co-localisation analysis of fluorescent signals representing FAM134B clusters and p62 or LC3B. Data are means ± s.d. of n = 10 cells per analysis.
