Igg mouse antibody

Bcl-2 mouse primary antibody (Cat.#610539, BD Bioscience, MA, USA and Cat.#658702 Biolegend, CA, USA). SOD1 mouse (Cat.#556360, BD Bioscience) and SOD2 rabbit (Cat.#06–984, Milipore, Sigma-Aldrich) primary antibody. COX5A mouse antibody (Cat.#ab110262, Abcam, Cambridge, UK and Santa Cruz). S70pBcl2 (Cat.#2827), S345pChk1 (Cat.#2348), γH2AX (Cat.#2577), S358pMLKL (Cat.#91689), MLKL (Cat.#14993T), Caspase-3 (Cat.#9662), GAPDH (Cat.#5174S) rabbit and Chk1 (Cat.#2360) mouse primary antibody (Cell Signaling, MA, USA). S87pBcl2 (Cat.#sc-16323) rabbit primary antibody and β-actin (Cat.#sc-47778) mouse primary antibody (Santa Cruz). T69pBcl2 (Cat.#PA5–36742) rabbit and BAX (Cat.#MA5–14003) mouse primary antibody (ThermoFisher Scientific, MA, USA). IgG rabbit (Cat.#3703, ProSci, CA, USA, Cat.#2729, Cell Signaling) and IgG mouse antibody (Cat.#sc-2025, Santa Cruz, TX, USA).

For IP, 25 million cells were washed with PBS and lysed using 340 µl Lysis Buffer (20 mmol/L TRIS, pH 7.5, 5 mmol/liter EGTA, 150 mmol/liter NaCl, 0.5% NP-40, phosphatase, and protease inhibitors 1×) on ice for 20 min, followed by centrifugation at 16,000 g at 4°C for 15 min. 40 µl of the supernatant was kept for the input fractions, and the rest was incubated overnight with 5 µg IgG mouse antibody (#sc-2025; Santa Cruz) or PGC1α antibody (#ST1202; Sigma-Aldrich) at 4°C overnight, followed by precipitation using magnetic DynaBead G (Invitrogen; prewashed twice with lysis buffer) for 1 h at 4°C. 40 µl of the supernatant was kept for the supernatant fraction, and the rest was discarded. Then, the beads were washed three times with lysis buffer and 50 µl of LDS 2× buffer, and 10 µl reducing agent was added. The samples were heated 4 min at 95°C, and 12 µl was used right away for immunoblots of total phosphoserine (#P5747; Sigma-Aldrich).