Tetra cell blot

For the preparation of cell lysates, the cells were twice washed with ice-cold PBS and lysed in lysis buffer (120 mM NaCl, 50 mM Tris–HCl [pH 7.2], 1% NP-40 [v/v], 1 mM EDTA, 6 mM EGTA, 6 mg/ml sodium pyrophosphate, 1× protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail [both Sigma–Aldrich]) for 30 min on ice. Lysates were cleared by centrifugation at 13,000 rpm/30 min/4 °C. Proteins were separated by Mops SDS-PAGE (39 (link)) and transferred to nitrocellulose membrane using the Tetra Cell-Blot (Bio-Rad) with 1× blotting buffer (20 mM Tris, 150 mM glycine, 20% methanol, and pH 8.3). Proteins were detected using rabbit anti-AGR2 (1:1000; K31 in house), mouse anti-AGR2 (1:500; Abnova), mouse anti-AGR2 (1:1000; AG3 4.1 in house), mouse anti-PDIA3, rabbit anti-PDIA6 (both 1:1000; Abcam), mouse anti-β-actin (1:1000; Sigma–Aldrich) as a loading control, and species-specific secondary horseradish peroxidase–coupled antibodies (1:1000; Dako). Densitometry analysis was performed using Quantity One software (Bio-Rad).

An amount of 8 µg of protein was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes for 2 h using the Tetra Cell-Blot (Biorad, Müchen, Germany) with 1× blotting buffer (20 mM Tris, 150 mM glycine, 20% methanol, pH 8.3). Western blot protein detection was performed with rabbit anti-Glutathione Reductase (GeneTex, GTX114199; 1:1000), mouse anti-β-Actin (R&D SYSTEM, MAB8929; 1:5000; Minneapolis, Minnesota, USA) antibodies in 5% dry milk/TBS/Tween, followed by species-specific secondary HRP-coupled antibody incubation (Invitrogen, Karlsruhe, Germany, 1:25,000). Protein bands were visualized using SuperSignal© Western Blot Enhancer (Thermo Scientific, Karlsruhe, Germany, 46640), SuperSignal© West Femto Maximum Sensitivity Substrate (Thermo Scientific, Karlsruhe, Germany, 34095) and AmershamHyperfilm ECL (GE Healthcare, Freiburg, Germany, 28906839) films.