Western blot was carried out as previously described [31 (link)]. Cells were lysed in RIPA buffer (with a cocktail of phosphatase and protease inhibitors) and solubilized in Laemmli buffer.
Amounts of 20 µg protein from cell lysates, determined by BCA Protein Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA), were separated by SDS–PAGE, blotted to nitrocellulose membrane, and probed with one of the following primary antibodies (dilution 1:400): anti-ERO1α (cat# 12007-1-AP, ProteinTech Group, Chicago, IL, USA), anti-GRP78/BiP (cat# ab21685, RRID: AB_2119834, Abcam, Cambridge, UK), anti-CHOP/GADD 153 (cat# ab11419, Abcam), anti-XBP1 (cat# ab37152, Abcam). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA; dilution 1:1000), developed by an ECL kit, acquired by ChemiDoc XRS (Bio-Rad Laboratories, Hercules, CA, USA), and digitized by Quantity One Imaging System (Bio-Rad). Equal loading was confirmed with anti-β-actin antibody (cat# A300-491A, Bethyl Laboratories, Montgomery, TX, USA).
Amounts of 20 µg protein from cell lysates, determined by BCA Protein Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA), were separated by SDS–PAGE, blotted to nitrocellulose membrane, and probed with one of the following primary antibodies (dilution 1:400): anti-ERO1α (cat# 12007-1-AP, ProteinTech Group, Chicago, IL, USA), anti-GRP78/BiP (cat# ab21685, RRID: AB_2119834, Abcam, Cambridge, UK), anti-CHOP/GADD 153 (cat# ab11419, Abcam), anti-XBP1 (cat# ab37152, Abcam). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Bethyl Laboratories, Montgomery, TX, USA; dilution 1:1000), developed by an ECL kit, acquired by ChemiDoc XRS (Bio-Rad Laboratories, Hercules, CA, USA), and digitized by Quantity One Imaging System (Bio-Rad). Equal loading was confirmed with anti-β-actin antibody (cat# A300-491A, Bethyl Laboratories, Montgomery, TX, USA).
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