Antibodies against PDI, BiP, and Hsp90 were purchased from Santa Cruz Biotechnology, Inc. Antibodies against ERp72 were obtained from StressGen Biotechnologies, antibodies against ubiquitin were purchased from Zymed Laboratories, and antibodies against IκBα were obtained from Biolegend. CT A and B antibodies and CT-GS were provided by the Lencer laboratory, the Tg antibodies were obtained from the Arvan laboratory, and the CT R192H mutant was provided by R. Holmes (University of Colorado, Boulder, CO). Hsp27, ERp57, and ERp29 antibodies were gifts from M. Welsh (University of Michigan, Ann Arbor, MI), S. High (University of Manchester, Manchester, England), and S. Mkrtchian (Karolinska Institutet, Stockholm, Sweden), respectively. CT, CTA, and PDI were purchased from Calbiochem. Mouse ERp72 cDNA was subcloned into pQE30, and the protein was purified using a Ni–nitrilotriacetic acid agarose column.
PDI-specific (5′-GACCTCCCCTTCAAAGTTGTT-3′) siRNA was synthesized by Ambion, and ERp72- (5′-CAAGCGUUCUCCUCCAAUUTT-3′) and ERp57-specific (5′-UGAAGGUGGCCGUGAAUUATT-3′) siRNAs were synthesized by Invitrogen. 10 nM duplexed siRNA was transfected into HeLa cells using Oligofectamine (Invitrogen) according to the manufacturer's protocol. Protein expression was assessed by SDS-PAGE and immunoblot analysis. Experiments were initiated 48 (ERp57) or 72 h (PDI and ERp72) after transfection.
Cells were incubated with 10 nM CT in HBSS at 37°C for 45 or 90 min. For permeabilization, 2 × 106 cells were resuspended in 100 μl of 0.04% digitonin in HCN buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 2 mM CaCl2, and protease inhibitors) with or without 10 mM NEM, incubated on ice for 10 min, and centrifuged at 16,000 g for 10 min. The supernatant was removed, and the pellet was washed with PBS and resuspended in 100 μl of the original buffer. Fractions were analyzed by nonreducing SDS-PAGE and immunoblot analysis. Where indicated, cells were treated with 2 μM MG132 for 90 min.
N-glycosylation of CTB
HeLa cells were incubated with 50 nM CT-GS in HBSS for 3 h at 4 or 37°C in the presence or absence of 5 μg/ml BFA. Cells were harvested in TN lysis buffer (1% Triton X-100, 1.75% n-octyl-B-/d-glucopyranoside, 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, and protease inhibitors), and the lysates were analyzed for the presence of glycosylated CTB.
A cAMP enzyme immunoassay system (GE Healthcare) was used to quantify cAMP synthesis induced by 10 nM CT or 50 μM forskolin in HBSS. Samples were assayed in duplicate, and the mean optical density was used to calculate the cAMP level/well. The cAMP response was determined by dividing the cAMP level in CT- or forskolin -treated cells by the cAMP level in unstimulated cells. Forskolin induced a sevenfold higher cAMP response in ERp72− cells than in wild-type cells. Results are reported as a percentage of the wild-type CT-induced cAMP response normalized to the forskolin-induced cAMP response.
Trypsin sensitivity assay
Purified CTA was incubated with 1 mM glutathione, 2 μg/ml BSA, 2 μg/ml PDI, or 0.2 μg/ml ERp72 for 30 min at 37°C. 100 or 300 μg/ml trypsin was added to the samples for 30 min at 4°C. Samples were analyzed by nonreducing SDS-PAGE followed by immunoblot analysis.
Accumulation of polyubiquitinated proteins
Cells were incubated with or without 20 μM MG132 for 30 min and lysed in a buffer containing 1% Triton-X, 10 mM NEM, 30 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, and protease inhibitors. Cleared lysates were analyzed by 4–20% SDS-PAGE followed by immunoblot analysis. To monitor ubiquitin-dependent degradation of IκBα, cells were treated with 10 ng/ml TNFα for 15 min, and the cell lysate was analyzed for the presence of IκBα.