Anti gclm

H9C2 cells with different treatment were lysed, quantified and subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Cat # 1,620,177, Bio-Rad, Hercules, CA, USA) which were incubated with 5% nonfat milk in TBST and followed by incubation of primary antibodies at 4°C overnight. Then the PVDF membranes were washed with TBST and incubated with secondary antibodies. Protein expression was detected using BeyoECL Moon (Cat # P0018FS, Beyotime) under a Tanon 5200 machine (Shanghai, China). The detailed primary and secondary antibodies were listed as below: anti-cleaved caspase 3 (Cat # E83-77, Abcam); anti-caspase 3 (Cat # ab32351, Abcam); anti-Bax (Cat # WL01637, Wanleibio, Shenyang, China); anti-Bcl-2 (Cat # WL01556, Wanleibio); anti-β-actin (Cat # WL01372, Wanleibio); anti-Nrf2 (Cat # WL02135, Wanleibio); anti-Sirt2 (Cat # ab211033, Abcam); anti-HO-1 (Cat # EP1391Y, Abcam); anti-GST (Cat # ab138491, Abcam); anti-NQO1 (Cat # 11,451-1-AP, Proteintech, Wuhan, China); anti-FOXO3a (Cat # 66,428-1-Ig, Proteintech); anti-GCLM (Cat # 14,241-1-AP, Proteintech); anti-Keap1 (Cat # 10,503-2-AP, Proteintech). IgG(H + L)(HRP-labeled Goat Anti-Rabbit IgG(H + L)) and IgG(H + L)(HRP-labeled Goat Anti-Mouse IgG(H + L)) were purchased from Beyotime (Beijing, China). All protein expressions were normalized by β-actin expression.

Cells were lysed in 1× Laemmli buffer and centrifuged at 10,000 g for 2 min through a Nucleo Spin filter (Machery Nagel). Lysates were boiled at 95 °C for 5 min, 20 μg of total protein were subjected to separation by a 10% SDS gel, transferred onto nitrocellulose membranes (Amersham Biosciences) and blocked with 5% milk powder in PBS Tween (0.1%) for 1 h. The following antibodies were used: anti-Nrf2 (monoclonal, 1:1000, Santa Cruz); anti-GCLC (rabbit, 1:1000, Bioworld Technologies); anti-GCLM (rabbit, 1:2000, Proteintech); anti-CYPOR (monoclonal, 1:500, Santa Cruz); HRP-conjugated anti mouse IgG (1:5000, Jackson Immuno Research); and HRP-conjugated anti-rabbit IgG (1:5000, Amersham). Protein bands were detected by a FUSION SL™ system (Peqlab, Erlangen, Germany) and quantified by ImaEva or ImageJ.