Rabbit anti caspase 3

To determine APN, LC3B, JNK/P-JNK, Akt/P-Akt, P62, and cleaved-caspase-3/pro-caspase-3 levels, proteins were extracted from the cells by suspension in radioimmunoprecipitation assay buffer. Samples were centrifuged at 12,000 rpm at 4°C for 30 min, and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the bicinchoninic acid protein assay kit (Sigma, St Louis, MO, USA). Protein (40 µg) was electrophoresed on a pre-cast bis-Tris polyacrylamide gel (8%~12%) and then transferred onto a polyvinylidene difluoride membrane. Membranes were blotted with rabbit anti-APN (1:1,000), rabbit anti-LC3B (1:500), rabbit anti-JNK (1:1,000), rabbit anti-p-JNK (1:1,000), rabbit anti-Akt (1:1,000), rabbit anti-p-Akt (1:1,000), mouse anti-P62 (1:1,000), rabbit anti-Caspase-3 (1:1,000) (all from Abcam, San Francisco, CA, USA), and mouse anti-actin (1:1,000; Proteintech, NY, USA), followed by horseradish peroxidase-conjugated secondary antibodies (1:5,000; ZsBio, Beijing, China). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000).

Western blot analysis was performed as described previously for evaluating the levels of Caspase-3 proteins (18 (link)). The samples (20 µg protein), as determined by using a bicinchoninic acid assay (Abcam), were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with the following primary antibodies: Rabbit anti-Caspase-3 (cat no. ab4051; 1:500; Abcam) antibody. GAPDH (cat no. G5262; 1:6,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used as a loading control. Following incubation with the primary antibodies for 1 h at room temperature, membranes were washed with TBS + 5% Tween-20 (TBST) and incubated with appropriate horseradish peroxidase-labeled secondary antibodies (cat no. sc2357; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature in 1% nonfat milk in TBST for 1 h at room temperature. Following two rinses and four washes with PBST, membranes were incubated with Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Healthcare Life Sciences, Shanghai, China) for 60 sec and exposed to autoradiography film for visualization of the bands. Results were quantified by Quantity One version 4.5 software (Bio-Rad Laboratories, Hercules, CA, USA). A total of 8 rabbits from each group were used.