Mouse anti gadph

Protein isolation and electrophoresis were performed as previously published [22 (link)]. Nitrocellulose or PVDF (Polyvinylidene fluoride) membranes were incubated with rabbit anti-AMOT 1:600 (provided by Dr. Lars Holmgren, Karolinska Institutet, Stockholm, Sweden), rabbit anti-AMOT 1:500 (Cell Signalling, #43130S), mouse anti-VE-cadherin 1:1000 (BD, #610252), or mouse anti-GADPH 1:1500 (Abcam, #ab8245), diluted in 2.5% nonfat dried milk/TBS-T (Tris Buffer Saline-0.05% Tween 20), at 4 °C o/n with gentle agitation. After washing with TBS-T, membranes were incubated with 25 ng/ml horseradish peroxidase-coupled secondary antibody (Pierce, #31430 or #31460) in 2.5% nonfat dried milk/TBS-T for 1 h at RT. HRP-conjugated proteins were visualized with Super Signal West-Pico Chemiluminescent Substrate (Thermo Fisher Scientific, #34087), followed by membrane autoradiography.

The cells were washed twice with PBS, lysed in a 100 μL protein lysis buffer (RIPA: PMSF = 100:1, Solarbio, Beijing, China), and centrifuged at 12,000 rpm at 4 °C for 15 min, and then the supernatant was frozen at −80 °C. The protein concentration was determined using BCA (Beyotime, Shanghai, China), in which equal amounts of protein (60 μg) were subjected to SDS-PAGE on a 10% or 12% gel. The separated proteins were electrophoretically transferred to PVDF membranes and 5% degreased milk was used asblocking agent for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-labeled antibodies for 1 h at room temperature. The signals were detected using an enhanced chemiluminescence reagent (Bio-Rad 170-5060, Hercules, CA, USA). The primary antibodies included mouse anti-GADPH (1:1000, ABCAm, Boston, MA, USA), rabbit anti-FTH1 (1:2000, ABCAm, Boston, MA, USA), rabbit anti-HO-1 (1:2000, CST, Danvers, MA, USA), rabbit anti-ACSL4 (1:10,000, ABCAm, Boston, MA, USA), and rabbit anti-GPX4 (1:2000, CST, Danvers, MA, USA).