Bcl 2 3498

DMEM, RPMI, trypsin-EDTA 0.25% and penicillin-streptomycin (PS) was purchased from GE Healthcare Life Sciences Hyclone Laboratories (Pittsburg, PA, USA). Fetal bovine serum (FBS) was received from Gibco (Thermo Fisher Scientific Inc., Walthem, MA, USA). ApoScan^TM annexin V-FITC was obtained from BioBud (Cat. No.: LS-02-100, Seongnam, Gyeonggi-do, Korea). MTT cell viability assay kit EZ-CYTOX was acquired from Do-GenBio Co. Ltd. (Seoul, Korea). PP2A activity kit was purchased from Millipore Corporation (Billerica, MA, USA). Antibodies against AKT (9272), pAKT (Thr308) (13038), Caspase-3 (9662), BAX (2772), and PARP (9542), and Bcl-2 (3498) were provided from Cell Signaling Technology (Danvers, MA, USA). β-actin (sc-47778), cytochrome-c (sc-13156), ERK 1/2 (sc-514302), pERK (Thr202/Tyr204) (sc-136521), and HRP-conjugated anti-mouse and anti-rabbit antibodies were received from Santa Cruz Biotechnology (Dallas, TX, USA). SK1 (ab71700) and SK1 (ab264042) antibodies were acquired from Abcam (Cambridge, UK). Protein marker, protease, and phosphatase inhibitor cocktail were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ECL Solution for western blotting imaging was received from Millipore Corporation (Burlington, MA, USA).

Roswell Park Memorial Institute-1640 (RPMI-1640), trypsin, and penicillin/streptomycin (P/S) were supplied from GE Healthcare Life Sciences Hyclone Laboratories (Pittsburg, PA, USA). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific Inc., Waltham, MA, USA). ApoScan^TM annexin V-FITC was obtained from BioBud (Seongnam, Gyeonggi-do, Korea). Cell viability assay kit EZ-CYTOX was received from Do-GenBio Co. Ltd. (Seoul, Korea). Antibodies against Caspase-3 (9662), BAX (2772), PARP (9542), and Bcl-2 (3498) were provided by Cell Signaling Technology (Danvers, MA, USA). β-actin (sc-47778) and HRP-conjugated anti-mouse and anti-rabbit antibodies were supplied from Santa Cruz Biotechnology (Dallas, TX, USA). SK1 (ab71700) and SK2 (ab264042) antibodies were acquired from Abcam (Cambridge, UK). Protein marker, protease, and phosphatase inhibitor cocktail were received from Thermo Fisher Scientific (Waltham, MA, USA). Electrochemiluminescence Solution for Western blotting imaging was offered by Millipore Corporation (Burlington, MA, USA). Alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) kits were purchased from Asan Corporation (Seoul, Korea).

The primary antibodies against B-cell lymphoma-2 (Bcl-2) (3498), Bcl-2-associated X (Bax) (2772), cleaved-caspase-3 (Asp175) (9661), and β-actin (4970) were all from Cell Signaling Technology (Danvers, MA, United States). Total protein was extracted from the left lower lobe using the ice-cold RIPA buffer (APPLYGEN). After protein was quantified with a BCA protein assay kit (Beyotime Institute of Biotechnology, China) and boiled with loading buffer (Solarbio, China), equal amounts of protein in each sample (100 μg) were prepared for electrophoresis on 8–12% SDS-PAGE and then electro-blotted onto PVDF membranes (Merck Millipore, United States). The membrane was incubated with 5% skim milk for 1 h at room temperature (RT), followed by incubation overnight with primary antibodies at 4°C (all antibodies were diluted following instructions). After the primary antibody was incubated, it was washed three times with TBST and then incubated with species-specific horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000) at RT for 1 h. After three washes with TBST, protein signal bands were visualized on a Chemidoc XRS (Bio-Rad, Marnes-la-Coquette, France) by an enhanced Chemiluminescence kit. Densitometry quantification of immunoblot signals was performed using ImageJ software. Each experiment was performed in triplicate.