Fig. 2: UBL3 does not control MHC II via altering synthesis, peptide loading, or UBL3 modification of MHC II.
a Analysis of Ii degradation and MHC II peptide loading in control MutuDCs expressing non-targeting hBim gRNA or ΔUbl3 MutuDCs. Cells were metabolically labeled for 30 min, washed, and cultured in a growth medium for the indicated time points before lysis. MHC II molecules were immunoprecipitated with JV1 rabbit antisera, or normal rabbit serum (NRS), and protein G-Sepharose. Samples were loaded before (NB) or after boiling (B) at 95 °C (b), subjected to denaturing SDS-PAGE, transferred onto PVDF membranes, and exposed to a storage phosphor screen. Sizes corresponding to free MHC II α and β, full-length invariant chain (Ii), the Ii spliced variant Iip41, the degradation intermediate Iip10, and SDS-stable complexes αβIip10, and αβ-peptide (pep), are indicated on the right. Data from one experiment. b ΔUbl3 MutuDCs or ΔUbl3 MutuDCs expressing Myc-UBL3 were lysed and post-nuclear supernatants (PNS) were subjected to immunoprecipiation (IP) with anti-Myc-Tag agarose (clone 9E10) or anti-MHC II antibody (M5/114) crosslinked to protein G-sepharose. Per lane, samples equivalent to 5 × 106 cells (Myc IP), 2.5 × 106 cells (MHC II IP), or 400,000 cells (PNS) were analyzed by non-denaturing SDS-PAGE and western blotting using antibodies against UBL3 (ab113820), MHC II β chain (JV2), Myc (71D10), and ubiquitin (P4D1). Shown are representative blots and quantification of relative ubiquitination, with bars displaying mean + 95% confidence intervals for four experiments.
