Figure 4. PDIA3 knockdown stabilizes... | Rockefeller University Press

Figure 4.

$PDIA3 knockdown stabilizes ubiquitinated antibody HC.(A) Cells that express mAb2 HC/LC were transfected with PDIA3 WT or the indicated mutants and subjected to subcellular fractionation. HC and HC fragment associated with the membrane fraction were analyzed by Western blotting. CGHA: PDIA3 C60A/C409A mutant that has the second cysteine residues of the CGHC motifs mutated to alanine residues, abolishing protease activity. AGHC: C57A/C406A mutant with the first cysteine of CGHC motifs mutated to alanine, abolishing isomerase activity while maintaining partial protease activity. (B) Cells were treated with the indicated cysteine protease inhibitors for 16 h, and proteins associated with the membrane fraction were analyzed. pHMB (25 µM). ALLN (10 µM): N-acetyl-Leu-Leu-Norleu-al or Calpain Inhibitor I. (C–F) Cells were treated with the indicated siRNA oligos. HC species that associated with the membrane fraction (C) or media (D) were analyzed by Western blotting. HC immunoprecipitated from the membrane fractions were analyzed for HC levels and BiP association (E) as well as HC ubiquitination (F). Arrowheads indicate where HC and HC fragment should be localized.$

PDIA3 knockdown stabilizes ubiquitinated antibody HC. (A) Cells that express mAb2 HC/LC were transfected with PDIA3 WT or the indicated mutants and subjected to subcellular fractionation. HC and HC fragment associated with the membrane fraction were analyzed by Western blotting. CGHA: PDIA3 C60A/C409A mutant that has the second cysteine residues of the CGHC motifs mutated to alanine residues, abolishing protease activity. AGHC: C57A/C406A mutant with the first cysteine of CGHC motifs mutated to alanine, abolishing isomerase activity while maintaining partial protease activity. (B) Cells were treated with the indicated cysteine protease inhibitors for 16 h, and proteins associated with the membrane fraction were analyzed. pHMB (25 µM). ALLN (10 µM): N-acetyl-Leu-Leu-Norleu-al or Calpain Inhibitor I. (C–F) Cells were treated with the indicated siRNA oligos. HC species that associated with the membrane fraction (C) or media (D) were analyzed by Western blotting. HC immunoprecipitated from the membrane fractions were analyzed for HC levels and BiP association (E) as well as HC ubiquitination (F). Arrowheads indicate where HC and HC fragment should be localized.

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