Gtx109683

Cells (1 × 10⁶) were lysed in RIPA lysis buffer (Solarbio Science & Technology Co., Ltd., Beijing, China) to collect total protein, and the protein concentration was examined using a bicinchoninic acid kit (Keygen Biotech Co., Ltd., Nanjing, Jiangsu, China) according to the instructions. An equal amount (60 μg) of protein sample was run on 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After being blocked in 5% not-fat milk for 1 h, the membranes were hybridized with the primary antibodies GAPDH (1: 10,000, ab181602, Abcam), GTSE1 (1:1000, GTX66223, GeneTex, CA, USA), Cyclin D1 (1:1000, #2978S, Cell Signaling Technology (CST), Beverly, MA, USA), Cyclin E1 (1:1000, #20808S, CST), proliferating cell nuclear antigen (PCNA; 1:1000, #13110S), cleaved caspase 3 (1:500, ab32042), Bax (1:1000, GTX109683, GeneTex), γH2AX (1:5000, ab81299), and DNA-PKcs (1:1000, ab32566) at 4 °C overnight, and then with HRP-conjugated secondary antibody (1:5000, ab205718, Abcam Inc., Cambridge, MA, USA) at room temperature for 1 h. The protein bands were developed using the ECL reagent (Pierce, Rockford, IL, USA).

After NaF treatment, total protein from hESCs was isolated using the mirVana™ miRNA Isolation kit (Life technologies). Protein lysates (50 μg/sample) were analyzed by western blot using primary antibodies, including mouse anti-human JNK (SC-7345, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human p-JNK (SC-293136, Santa Cruz Biotechnology), rabbit anti-human ERK (GTX59618, Genetex, Irvine, CA), rabbit anti-human p-ERK (2219–1, Epitomics, Burlingame, CA), rabbit anti-human BCL-2 (GTX100064, Genetex), rabbit anti-human BAX (GTX109683, Genetex) and mouse anti-human GAPDH (TA08, ZSGB-BIO, Beijing, China). The horseradish peroxidase (HRP)–conjugated donkey anti-mouse IgG (ZB5305, ZSGB-BIO) or donkey anti-rabbit IgG (ZB5301, ZSGB-BIO) were used as secondary antibodies. SuperSignal West Pico Trial Kit (Thermo Scientific, Rockford, IL) was applied for protein detection. The intensity of individual bands was quantified using the ImageJ densitometry software.

H22 cells (3 × 10⁵cells/hole) in a logarithmic growth phase were plated in a 6-well plate and were incubated with B. coagulans MZY531 (MOI = 0, 50, 100) and 5-FU (100 µg/mL). After 24 h, the whole cell extract was prepared with RIPA buffer containing 1 mm PMSF. The protein concentration was detected by the BCA method, separated by SDS-PAGE electrophoresis, and transferred to the PVDF membrane. Western blot analysis was performed using the following primary anti-rabbit monoclonal antibodies: β-actin (bsm-52846R, BIOSS), PI3K (bs-10657R, BIOSS), p-PI3K (bs-5570R, BIOSS), AKT (bs-0115R, BIOSS), p-AKT (bs-0876R, BIOSS), mTOR (bsm-54471R, BIOSS), p-mTOR (bs-5331R, BIOSS), and Bax (GTX109683, GeneTex), caspase-3 (GTX110543, GeneTex) and Bcl-2 (GTX100064, GeneTex). The membrane was incubated with anti-rabbit second antibody conjugated with horseradish peroxidase at 37 ℃ for 1 h. the strips were quantified using image quant Las 4000 (Fuji film, Tokyo, Japan), and β-actin was used as a loading control.