Fig. 3: CK2 activity positively impacts FAM134-driven ER-phagy.

A Schematic representation of the mass spectrometry experimental procedure to identify FAM134s phosphorylation sites under basal (DMSO) or induced (Torin1) conditions. Created with BioRender.com. B Schematic representation of FAM134B or FAM134C proteins highlighting the reticulon homology domain (RHD; white) comprising two transmembrane domains (TM1/2 and TM3/4; blue) flanked by membrane associated linker regions (green), the conserved LC3 interacting motif (LIR), as well as sites of phosphorylation events. C Representative blot image of in vitro phosphorylation of full-length GST-FAM134B by purified CK2 and corresponding Coomassie-stained gel (lower panel). Anti-GST antibody was used to detect GST-FAM134B (upper panel). n = 3 biological replicates. D Schematic three-dimensional representation of FAM134B-RHD highlighting serine 153 phosphorylated in vitro by CK2. E, F Left panels: representative blot images validating the stable expression of FAM134 WT or phospho-mutant proteins under DOX induction in the background of U2OS cells expressing ER-phagy reporter ssRFP-GFP-KDEL. Right panels: ER-phagy flux in U2OS cells expressing FAM134s WT or phospho-mutants and ER-phagy reporter ssRFP-GFP-KDEL, represented by the RFP/GFP ratio of total integrated intensities in basal (DMSO) or induced (Torin1) conditions. These data represent averaged data obtained from n = 3 individual wells via the IncuCyte® S3, each view containing >100 cells. Data are mean ± SD, [au] arbitrary unit. Representative blot image (G) and the relative bar plot (H) showing Collagen I accumulation in WT MEFs, Fam134b KO or cells reconstituted with FAM134B WT and phospho-mutant (S149,151,153A). Representative blot image (I) and the relative bar plot (J) showing Collagen I accumulation in WT MEFs, Fam134c KO or cells reconstituted with FAM134C WT and phospho-mutant (S258A). Data information: GAPDH was used as a reference for ratio calculation. The statistical significance was estimated by one-way ANOVA. n = 4 biological replicates for (G, H) and n = 3 biological replicates for (I, J). Data are mean ± SD, [au] arbitrary unit. Source data are provided as source data file.