Fig. 3.

Full-length MHC I HC is preferentially ubiquitinated on ER luminal residues. (A) Soluble MHC I HC is ubiquitinated in β2m-depleted cells. HeLa shβ2m cells expressing wild-type (wt) or soluble (sol) HA-HLA-A2 were incubated with or without 50 μM MG132 for 5 h, before SDS lysis, immunoprecipitation with anti-HA, and immunoblot for polyubiquitin and HA. (B and C) Full-length MHC I HC is preferentially ubiquitinated on ER luminal residues in β2m-depleted cells. Lysates from HeLa cells expressing HA-HLA-A2 TEV303 with β2m shRNA (MG132-treated), K3 viral E3 ligase or alone (control), were immunoprecipitated with anti–HA-agarose beads. On-bead incubation with TEV protease released the MHC I C terminus into the supernatant, and the residual HA-tagged N-terminal (luminal) fragment was eluted from the beads. Bead and supernatant fractions were probed for ubiquitin, HA, and MHC I C terminus (R.A3e7).