Criterion bis tris gel

For the inducible cell line, 8 × 10⁶ HEK293^doxIRE1β cells were seeded in 100-mm dishes. The next day, IRE1β-FLAG expression was induced with 100 ng/ml DOX for 24 h. For transient overexpression, 8.2 × 10⁶ HEK293T cells were seeded in 100-mm dishes. The next day, IRE1β-FLAG plasmid was transfected using PEI at a 1:5 DNA/PEI ratio (5 µg DNA). Cells were collected after 24 h. Cells were lysed in lysis buffer (0.1% NP-40, 10% glycerol, 250 mM NaCl, 20 mM Hepes, pH 7.9, and 1 mM EDTA) with protease and phosphatase inhibitors. Samples were incubated with 10 µl prewashed Dynabeads T1 (Invitrogen) that were either prebound with BioM2 anti-FLAG (Sigma; 1 µl antibody/10 µl beads) or no antibody for 1 h at 4°C. After washing with lysis buffer, bound proteins were eluted directly in sample buffer and samples were separated on a 4–12% Criterion Bis-Tris gel (BioRad). Proteins were transferred to polyvinylidene difluoride membrane (Immobilon-FL; Millipore), and incubate with anti-IRE1a (1/1,000; 14C10; Cell Signaling Technology), M2 anti-FLAG (1/1,000; Sigma), and anti-tubulin (1/2,000; Sigma). Proteins were revealed with anti-mouse and anti-rabbit HRP-conjugated antibodies (1/15,000; Jackson Immuno AffiniPure).