Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody

Briefly, Cells treated with different antibodies were collected and lysed in RIPA buffer (beyotime), containing protease inhibitor cocktail (Abcam), and incubated on ice for 1 h with an interval vortex. The lysates underwent centrifugation at 12,000 rpm for 15 min at 4 °C. The total protein concentrations in the resulting supernatant fractions were determined using a BCA protein assay kit (beyotime). Then the equal amounts of protein samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membranes. After blocking with 5% milk, the membranes were blotted with LC3 polyclonal antibody (Proteintech), Rabbit PAb (1:2000) and GAPDH antibody (Servicebio), Rabbit PAb (1:3000) incubated in blocking solution overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:3000) (Proteintech) and visualization with Chemiluminescent Reagent (Bio-Rad, USA).

Annexin V-enhanced green fluorescent protein (EGFP)/propidium iodide (PI) apoptosis detection kit, cell cycle detection kit, DAPI, bicinchoninic acid assay kit, and cell lysis assay kit containing protease and phosphatase inhibitors were purchased from Nanjing Keygen Biotech, Co., Ltd. 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and trypan blue dye were purchased from Beyotime Institute of Biotechnology. In situ cell death detection TUNEL kit was purchased from Roche Diagnostics GmbH. 3-Methyladenine (3-MA) and rapamycin were purchased from Selleck Chemicals. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (Merck KGaA). Cycloheximide (CHX) was purchased from MedChem Express. Rabbit monoclonal antibodies against β-actin (cat. no. 4970), γ-H2AX (cat. no. 9718), caspase-3 (cat. no. 9662), cleaved caspase-3 (cat. no. 9664), LC3 (cat. no. 3868), CHOP (cat. no. 5554) and Ki-67 (cat. no. 9027) were obtained from Cell Signaling Technology, Inc. Rabbit polyclonal antibodies against p62 (cat. no. 18420) and Grp78/Bip (cat. no. 11587) were obtained from ProteinTech Group, Inc. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. G-21234) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (cat. no. A-11008) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.).