Protein a g

Glucose, streptozocin (STZ), and Evans blue were purchased from Sigma-Aldrich (St. Louis, MO, USA), while advanced glycosylation end products (AGEs) were acquired from BioVision (Palo Alto, CA, USA). Tunicamycin (TUN) was obtained from Abcam (Cambridge, CA, USA), while 4-phenylbutyric acid (4-PBA), BAPTA-AM, and Protein A/G magnetic beads were purchased from MedChemExpress (Lowell, NJ, USA). Cell counting kit-8 (CCK8), JC-1 assay kit, Calcein/PI assay kit, MPTP assay kit, Mito-Tracker Green, ER-Tracker Red, and a mitochondria isolation kit were purchased from Beyotime (Nantong, China). On the other hand, the TUNEL assay kits were purchased from Roche (Basel, Switzerland). MitoSOX™ Red, DAPI, ECL kits, and goat anti-mouse/rabbit IgG (H+L, Alexa Fluor 555/488) were acquired from Thermo Fisher (Waltham, CA, USA). We used primary antibodies including GRP75, Cyt c, Bcl-xl, Bax, cleaved caspase-3, and CHOP (Cell Signaling Technology, Boston, MA, USA); 4-HNE and Brn3a (Abcam, Cambridge, CA, USA); IP3R1, VDAC1, VEGF, 8-OHDG, and 3-NT (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and COX IV, Bcl-2, and β-actin (Proteintech, Wuhan, China). More detailed information about the materials and reagents is offered in Table S1.

Cells were lysed in Co-IP buffer (pH 7.5, 1% Triton X-100, 150 mM NaCl, protease inhibitor cocktail, 20 mM Tris, and 1 mM EDTA) on ice for 20 min. Cell supernatant (200 μl) was collected and then incubated with control IgG, primary antibodies against Flag (4 μg, BioLegend, USA, # 637303) or PirB (4 μg, Thermo Fisher Scientific, MA, USA, Cat#MA5-24049) with gentle rocking at 4 °C overnight followed by 1-h incubation with Protein A/G (MedChem Express, NJ, USA, #HY-K0202) at 4 °C. After three washes with co-IP lysis buffer, protein samples were collected BCby boiling in 3× SDS loading buffer and subjected to western blotting.