Fig. 1: RNF185/MBRL-deficient cells display elevated levels of the MHC-I chaperone TPSN.

a Schematic overview of the workflow for substrate identification in MBRL-deficient mouse astrocytes. Primary cortical astrocytes from 5 parental and 5 MBRL KO P1–P3 mouse pups were cultured ex vivo for 21 days. Astrocytes were harvested, lysed and protein extracts digested. The resulting peptide samples were labelled with TMT-10-plex reagent and analysed by LC-MS/MS. Moderated t-tests, with patient accounted for in the linear model, were performed using Limma, where proteins with p-value < 0.05 were considered as statistically significant. b Identification of proteins increased and decreased in MBRL KO mouse astrocytes compared to wildtype astrocytes. Volcano plot showing the relation of log2 fold-change and -log10 p-value of protein levels in MBRL KO vs. wildtype mouse astrocytes. Proteins that are significantly decreased in MBRL KO astrocytes are on the left and labelled in Orange. Proteins that are significantly increased in MBRL KO astrocytes are on the right and labelled in Blue. Astrocytes from 5 animals were analysed for each condition. c Validation of the proteins identified in b. Protein extracts from wildtype and MBRL KO mouse astrocytes were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. Of note, the anti-RNF185 antibody cross-reacts with RNF5, as indicated. The asterisk (*) indicates a non-specific background band. d, e TPSN protein levels are increased in RNF185 and MBRL KO human iPSCs and HEK293 cells. Extracts from parental, RNF185 and MBRL KO iPSCs (d) and HEK293 Flp-In T-Rex cells (e) were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. The asterisk (*) indicates a non-specific background band.