Anti cdc2

PL (S7551) was purchased from Selleck Chemical (Shanghai, China), the purity of PL is 99.33%. N-Acetylcysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). PI was purchased from BD Pharmingen (Franklin Lakes, NJ). Hoechst stain and DCFH-DA were purchased from Beyotime Biotechnology (Nantong, China). Anti-Cdc2, anti-Bcl-2, anti-Bax, anti-CyclinB1, anti-TrxR1, and anti-Ki67 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-ATF-4, anti-EIF2α, anti-CHOP, and anticleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA). HRP-conjugated secondary antibodies were also obtained from Cell Signaling Technology.

The samples were then loaded onto 10% SDS–polyacrylamide gels. After transferring onto PVDF membranes, immunoblotting was performed using anti-Cdc2 (Santa Cruz Biotechnology, sc-53217) antibody, anti-phospho Cdc2 p34 (Tyr 15) (Santa Cruz Biotechnology, sc-7989) antibody, anti-phospho p38 [Cell Signaling Technology, 9211S; for phospho (p)-Sty1] or anti-Sty1 (lab stock) antibodies at 1:1000 dilution (or 1:2500 dilution for anti-Sty1). Immunoblots were developed using AdvanstaWesternBright™ ECL reagent (K-12045-D50).

The samples were then loaded onto 10% SDS–polyacrylamide gels. After transferring onto PVDF membranes, immunoblotting was performed using anti-Cdc2 (Santa Cruz Biotechnology, sc-53217) antibody, anti-phospho Cdc2 p34 (Tyr 15) (Santa Cruz Biotechnology, sc-7989) antibody, anti-phospho p38 [Cell Signaling Technology, 9211S; for phospho (p)-Sty1] or anti-Sty1 (lab stock) antibodies at 1:1000 dilution (or 1:2500 dilution for anti-Sty1). Immunoblots were developed using AdvanstaWesternBright™ ECL reagent (K-12045-D50).

Human NSCLC cell line A549 and H460 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetalbovine serum (Gibco, Eggenstein, Germany), 100 units/mL penicillin, and 100 ug/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO₂ at 37 °C. Antibodies including anti-MDM2 (sc-965, 1:200), anti-Cdc2 (sc-54, 1:200), anti-Cdc25C (sc-13138, 1:200) anti-Cyclin B1 (sc-245, 1:200), anti-Bcl-2 (sc-7382, 1:200), anti-Bcl-xL (sc-7382, 1:200), anti-cleaved PARP-1 (sc-56196, 1:200), anti-GAPDH (sc-293335, 1:1000), goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-ATF4 (#11815, 1:1000), anti-p-EIF2α (#3398, 1:1000) and anti-EIF2α (#9722, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), Licochalcone A and DMSO (dimethyl sulfoxide) were purchased from Sigma (St. Louis, MO, USA); FITC Annexin V apoptosis Detection Kit I and PI (propidium iodide) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA).

Human gastric cancer cell lines SGC-7901, BGC- 823 and KATO III were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 ug/ml streptomycin in a humidified cell incubator with an atmosphere of 5% CO2 at 37°C. FITC Annexin V apoptosis Detection Kit I and propidium iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and JC-1 were purchased from Invitrogen (Carlsbad, CA). Antibodies including anti-Cdc2, anti-XBP-1, a'nti-Bcl-2, anti-Bax, anti-Cyclin B1, anti-cleaved PARP, anti-MDM-2, anti-p53, anti-TrxR1, anti-Ki67, anti-GAPDH, goat anti-mouse IgG-HRP, donkey anti-rabbit IgG-HRP and donkey anti-goat IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-ATF-4, anti-p-EIF2α, anti-CHOP, anti-Cle-caspase3, were purchased from Cell Signaling Technology (Danvers, MA).