Anti ire1α antibody

NCI-H929 cells were treated with 10 μg/ml tunicamycin for 4 hours. Cells were lysed in buffer containing 1% Triton X-100; 10 mM Tris pH7.6; 10 mM EDTA; 150 mM NaCl and twice standard concentrations of PhosStop and complete protease inhibitors (Roche). Lysates were spun at 12000 × g for 10 minutes to remove nuclei. Supernatants were transferred to fresh tubes and incubated overnight with anti-IRE1α antibody (Cell Signaling Technology, Danvers, MA, USA). Antibody:IRE1α complexes were captured with protein A/G magnetic beads (Thermo Fisher Scientific, Hemel Hempstead, UK) and washed five times in lysis buffer before elution into reducing Laemmli sample buffer. Proteins were separated by SDS-PAGE and stained with coomassie for mass spectrometry.

Sulfonation was performed as was previously reported [33 (link)]. Total thoracic aorta tissues and HUVECs (~500 μg) were prepared using lysis buffer (Cell Signaling Technologies) containing protease and phosphatase inhibitors (Sigma-Aldrich). Immunoprecipitation was performed with anti-cysteine-sulfonate (#ab176487, Abcam), followed by an anti-IRE1α antibody (#3294, Cell Signaling) to detect IRE1α sulfonation. Protein A/G Sepharose beads (Sigma-Aldrich) were added and incubated for 1 h. Immunoprecipitates were washed 5 times with PBS-T buffer or PBS, separated using SDS-PAGE, and probed with specific antibodies.

Palmitic acid (PA) was purchased from Aladdin (Shanghai, China). Oleic acid (OA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), and stearic acid (SA) were purchased from Sigma (Shanghai, China). Bovine serum albumin (BSA) was purchased from Solarbio (Beijing, China). A Cell Counting Kit-8 (CCK-8) was purchased from Beyotime (Shanghai, China). Anti-Cleaved caspase-3, anti-IRE1α antibody, anti-p-eif2a antibody, and anti-BAX antibody were purchased from Cell Signaling Technology (Hong kong, China). Donkey Anti-Mouse IgG H&L (Alexa Fluor^® 488), Donkey Anti-Rabbit IgG H&L (DyLight^® 550), and anti-BCL-2 antibody were purchased from Abcam (Cambridge, UK). Anti-ATF6 antibody and anti-CHOP antibody were purchased from Affinity bioscience (Jiangsu, China). Anti-actin antibody was purchased from ABclonal (Shanghai, China). Anti-BIP antibody and anti-glucagon antibody was purchased from absin (Shanghai, China). Anti-Insulin antibody and anti-ATF4 antibody were purchased from Huaan (Hangzhou, China). Anti-XBP-1 antibody and HRP labelled goat anti-rabbit IgG were purchased from Wanlei (Shenyang, China). TRIizol reagent was purchased from Ambion (TEXAS, USA). A high-capacity cDNA synthesis kit was purchased from Vazyme (Nanjing, China).