Rabbit anti bax

Human SCC cell lines, NCI-H520 and SK-MES-1, and human SCLC cell lines, DMS114 and NCI-H2227, were obtained from the American Type Culture Collection (Manassas, VA, USA). Antibodies were purchased from commercial sources, including rabbit anti-CUL4B antibody (1:50; Novus Biologicals, Littleton, CO, USA), rabbit anti-CUL4A (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-FOXO3A (1:1,000; Cell Signaling Technology), rabbit anti-p-FOXO3A (1:1,000; Cell Signaling Technology), rabbit anti-cyclin E1 (1:1,000; Abcam, Cambridge, UK), rabbit anti-ERK (1:1,000; Abcam), rabbit anti-p-ERK (1:1,000; Abcam), rabbit anti-P21 (1:1,000; Proteintech, Rosemont, IL, USA), rabbit anti-CUL4B (1:1,000; Proteintech), rabbit anti-BCL-2 (1:1,000; Proteintech), rabbit anti-BAX (1:1,000; Proteintech), rabbit anti-caspase 3 (1:1,000; Proteintech), rabbit anti-UBC12 (1:1,000; Proteintech), and mouse anti-β-actin (1:1,000; Sigma-Aldrich, St. Louis, MO, USA). Si-RNAs were purchased from RiboBio (Guangzhou, China), including si-FOXO3A and si-UBC12. MG132, MLN4924, and PD98059 were purchased from Selleck Chemicals (Houston, TX, USA). Cycloheximide (CHX) was purchased from Absin (Shanghai, China).

The cells in each group were lysed in M-PER cell lysis buffer (Thermo Fisher Scientific, USA). The total protein of each group was collected and quantified measuring with a BCA total protein kit (Thermo Fisher Scientific, USA). Proteins of the same volume were separated by SDS-PAGE. Then, the proteins were transferred to the semidry membrane. Subsequently, membranes were blocked in 5% BSA for 1 hour at room temperature and incubated with β-actin (1 : 2500, Proteintech), rabbit anti-BAX (1 : 250, Proteintech), rabbit anti-Bcl-2 (1 : 1000, Abcam), rabbit anti-JAK2 (1 : 1000, Cell Signaling Technology), and rabbit anti-STAT3 (1 : 1000, Cell Signaling Technology) antibodies at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies labeled with horseradish peroxidase (HRP) for 2 hour. The membrane was exposed to the developing solution of electrogenerated chemiluminescence (ECL) (Advansta, CA, USA). The optical density of protein bands was analyzed by ImageJ image analysis software.

Total protein was extracted using RIPA lysis buffer containing protease inhibitors (APExBIO Technology, Houston, USA). Protein concentrations were determined using BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Then 10% SDS-PAGE gel electrophoresis was carried out using 50 μg of protein from each sample. After gel transfer, PVDF membranes were incubated with primary antibodies including rabbit anti-Bax (1:1,000, Proteintech, Wuhan, China), Bcl-2 (1:1,000, Proteintech, Wuhan, China), survivin (1:1,000, Proteintech, Wuhan, China), cleaved caspase-3 (1:1,000, Proteintech, Wuhan, China), cleaved caspase-9 (1:1,000, Proteintech, Wuhan, China), p21 (1:1,000, Proteintech, Wuhan, China), cyclin D1 (1:1,000, Proteintech, Wuhan, China), IRS1(1:1,000, Affinity, USA), p-PI3K (1:1,000, Affinity, USA), p-AKT (1:1,000, Affinity, USA), PI3K (1:1,000, Affinity, USA), and AKT (1:1,000, Affinity, USA) overnight at 4°C. After washing, membranes were further incubated with fluorochrome-labeled anti-rabbit IgG secondary antibody (1:10,000, Proteintech, Wuhan, China) for 1 h at room temperature. The membranes were imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), which was used to normalize relative expression level of each protein to GAPDH.

Western blot analyses were performed as described in a previously published paper [28 (link)]. Total proteins were extracted from SGC-7901 and HGC-27 cells 48 hours after transfection by the radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (PIC), and the protein concentration was measured using the bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). The protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, U.S.A.). Membranes were blocked with 5% milk and incubated with the following primary antibodies: rabbit anti-BAX (catalog# 50599-2-Ig, 1 : 1,000; Proteintech, Wuhan, China), mouse anti-Caspase3 (catalog # 66470-2-Ig, 1 : 1,000; Proteintech, Wuhan, China), mouse anti-HuR (catalog# sc-5261, 1 : 1,000; Santa Cruz, Dallas, U.S.A.), and mouse anti-VINCULIN (catalog# ab129002, 1 : 1,000; Abcam, Cambridge, UK), and then incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (catalog# 1460 or 31430, both diluted to 1 : 10,000; Thermo, Waltham, U.S.A.) for 1 hour at room temperature (RT). Finally, protein bands were detected by chemiluminescence (ECL) reagents. The intensity of the bands was quantified using ImageJ software (National Institutes of Health, Maryland, U.S.A).

RIPA buffer containing PMSF and phosphatase inhibitors was used to extract the proteins of renal tissue and HK2 cells protein (all from Beyotime, China). According to the manufacturer's protocol, the protein concentration was determined by the BCA protein assay kit. The protein samples of each group were separated by SDS-PAGE and transferred to PVDF membranes (MILI Bloomberg, Massachusetts). The PVDF membranes were sealed with rapid blocking solution at room temperature. The primary antibodies included rabbit anti PICK1 (1 : 500, protein tech,) rabbit anti Cleaved Caspase-3, rabbit anti Bax, and rabbit anti BCL-2 (1 : 500, Proteintech, China), rabbit anti ASK1 (1 : 500,Proteintech, China), rabbit anti p38MAPK (1 : 500, Proteintech, China), and rabbit anti GAPDH (1 : 8000; Proteintech, China). Using the HRP-labeled secondary antibody (1 : 3000 dilution) to incubate the membrane at 37°C for 1 hour. Finally, the fusion imaging system is used to detect the relative densities of the bands.