Anti bcl 2

The protein expressions of ETS1, MDR1, and apoptotic proteins were measured by western blot. Total cell lysates were generated by RIPA lysis buffer (Beyotime, China) and quantified with a BCA kit (Thermo Fisher Scientific, USA). An equal amount of total protein was resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and then transferred to PVDF membrane (Bio-Rad, USA). The membranes were then blocked with 5% non-fat milk at 37°C for 1 h and incubated with the following primary antibodies: Anti-ETS1 (sc55581), anti-MDR1 (sc55510), anti-Bcl-2 (sc7382), anti-Bax (sc7480), and anti-GAPDH (sc47724; Santa Cruz Biotechnology, USA). Finally, the membranes were incubated with secondary antibodies and visualized by an electrochemiluminescence system (Amersham, USA).

TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The First Strand cDNA Synthesis kit and SYBR Premix Taq were from Takara (Dalian, Liaoning, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), bromodeoxyuridine (BrdU) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). DAPI, BCA protein assay and ECL Plus kits were obtained from Beyotime Institute of Biotechnology (Beijing, China). BD BioCoat Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA).
The primary antibodies against human SKA1 and cleaved caspase-3 were obtained from Abcam (Cambridge, MA, USA). Anti-Bcl-2, anti-Bax, anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt, anti-p21, anti-cyclin D1 and anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Biotinylated- and Cy3-conjugated anti-rabbit secondary antibodies were purchased form Boster (Wuhan, Hubei, China).

Western blotting was performed as described previously (Xie et al., 2017 (link)) with slight modifications. Cell pellets were resuspended in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1mM EDTA pH 8.0, and protease cocktail inhibitor). Soluble extracts were prepared by centrifugation (14,000 × g for 20 min at 4°C). Cell lysates were separated by 6–15% SDS-PAGE and then transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked for 1 h with 3% dried skim milk in TBST (50 mM Tris pH 8.0, 150 mM NaCl, and 0.5% Tween 20) and incubated with the following primary antibodies, anti-HA (Santa Cruz, sc-805), anti-FLAG (Sigma Aldrich, F1804), anti-MYC (Cell Signaling, #2276), anti-BCL-xL (Cell Signaling, #2764), anti-MCL-1 (Santa Cruz, sc-819), anti-ACTIN (Santa Cruz, sc-47778), anti-GAPDH (Santa Cruz, sc-32233), anti-BCL2 (Santa Cruz, sc-7382), anti-BAX (Santa Cruz, sc-493), and anti-PARP1 (Santa Cruz, sc-7150). After incubation with primary antibodies, the membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (GeneDEPOT, USA). Protein expression was detected by enhanced chemiluminescence (ECL) reagents and LAS-3000 image analyzer (Fujifilm, Japan).