Phenylmethanesulphonyl fluoride pmsf

The tissue or cells are lysed in the cell lysis buffer for Western and IP (Beyotime, Shanghai, China) and phenylmethanesulphonyl fluoride (PMSF) (Beyotime, Shanghai, China). The lysate was centrifuged at 12,000× g for 20 min at 4 °C, then 80 μL of supernatant was added to 20 μL of 5×loading buffer and boiled at 100 °C for 10 min. The proteins were analyzed by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA), and blocked with 5% skim milk containing 0.05% Tween (TBS-T). After washing three times in TBS-T, the membrane was incubated with anti-PKC-δ-like antibody (1 μg/mL) or anti-flag antibody (Abcam, Cambridge, UK) (1 μg/mL) at 4 °C TBS-T and 5% skim milk overnight. After washing, the membrane was incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) in TBS-T and 5% skim milk at 25 °C for 1 h. Signals were displayed using Enhanced Chemiluminescence (ECL) kits (Sangon Biotech, Shanghai, China). Rabbit anti-β-actin (Sangon Biotech, Shanghai, China) is used for standardized protein quality in each lane.

Antithrombin, rapamycin, A6730, Evans blue, and triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-beclin-1 (1:1000), anti-GAPDH (1:5000), anti-protein kinase B (Akt; 1:1000), anti-phosphorylated (p)-Akt (Ser473,1:1000), anti-autophagy protein microtubule-associated protein 1 light chain-3B (LC3B; 1:1000), anti-B-cell lymphoma 2 (Bcl-2; 1:1000), anti-Bcl-2 associated x (Bax; 1:1000), and anti-p62 (1:1000) antibodies were obtained from either Cell Signaling Technology (Danvers, MA) or Abcam (Cambridge, UK). Phenylmethane sulphonyl fluoride (PMSF), the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit, and the bicinchoninic acid protein quantification kit were obtained from Beyotime (Shanghai, China). The creatine kinase (CK) assay kit was purchased from the Jiancheng Bioengineering Institute (Nanjing, China). Goat anti-rabbit secondary antibodies were purchased from Biosharp (Guangzhou, China).