UBR4 and UBR5 ubiquitinate antibody HC and target it for degradation. (A) Knockdown of UBR4 and UBR5 by siRNA transfection. UBR4/5: UBR4 and UBR5. (B) Intracellular levels of HC in HC-expressing cells were determined after cells were treated with the indicated UBR siRNA oligos for 4 d. (C) Levels of full-length HC and HC fragment in the membrane fraction of HC-expressing cells treated with the indicated UBR siRNA oligos. (D) Pools of mAb2 HC/LC- or HC only–expressing cells were treated with control siRNA or both UBR4 and UBR5 specific siRNA oligos. Levels of full-length HC and HC fragment in both the intracellular membrane fraction and in the media (harvested cell culture fluid or HCCF) were analyzed by Western blotting. Calnexin (an ER protein) was used as loading control of intracellular membrane fractions. (E) Cells expressing mAb4 HC/LC were transfected with indicated siRNA(s) and subjected to Western blot analysis for HC and actin. Relative HC/actin band intensity ratios have been quantified for each sample and normalized to the control. (F) Cells that stably express Flag-tagged mAb2 HC and HA-tagged WT or K48-specific mutant ubiquitin were transfected with UBR4/5 or control siRNA oligos followed by MG132 treatment. HC molecules in these cells were then immunoprecipitated. The levels of HA-Ub modifications were determined by Western blotting. (G) Cells that stably express Flag-tagged mAb1 HC and HA-tagged K48, K63, or K11-specific ubiquitin mutants were treated as in F. HC molecules were immunoprecipitated, and HA-Ub modifications were determined by Western blotting. K48, K63, or K11-specific mutants of ubiquitin have all lysine residues mutated to alanine except for the indicated lysine residue. hIgG: human IgG. Arrows indicate the expected migration positions of HC and HC fragment bands on SDS-PAGE.