P bcl 2

HCT116 and SW620 cells were lysed by cell lysis solution (1× PBS, 1% Nonidet P40, and 1 mM EDTA) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein were diluted in 2× sodium dodecyl sulfate (SDS) loading dye, separated on 8–15% SDS-polyacrylamide (SDS-PAGE) gels, and transferred onto nitro-cellulose membranes (GE Healthcare Life Sciences, Boston, MA, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were probed overnight with primary antibodies at 4 °C. Anti-O-GlcNAc antibody was purchased from Sigma-Aldrich. An anti-PARP antibody was purchased from Invitrogen. Anti-cleaved caspase-3, IRE1α, P-eIF2α, eIF2α, CHOP, P-AMPK, AMPK, P-ULK, P-mTOR, mTOR, P-p70 S6K, p70 S6K, ATG5, and LC3B antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ULK, PERK, ATF6, ATF4, P-JNK, JNK, Bax, P-Bcl2, Bcl2, p62, GFAT, caspase-8, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G secondary antibodies at room temperature for 30 min. Immunoreactive bands were developed using an ECL Plus Detection System (GE Healthcare Life Sciences) and the bands were quantified using ImageJ software (NIH, USA).

Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). The following antibodies were purchased: Rac1 antibody (BD Biosciences, Franklin Lakes, NJ, USA); c-Jun N-terminal kinase (JNK), p-JNK, p-p38, p38, p-ERK, ERK, p-PKC, PKC, p-c-Src, c-Src, p-NF-κBp65, NF-κBp65, p-c-Jun, c-Jun, Bax, p-Bcl-2, Bcl-2, caveolin-1, cleaved caspase-3, caspase-9, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); Bax (6A7) monoclonal antibody (Thermo Fisher Scientific, Rockford, IL, USA); LC-3, NCF-1 and Beclin-1 antibodies (Novus Biologicals, Littleton, CO, USA). VvhA-specific antibody was acquired from Professor Sang Ho Choi (Seoul National University, Seoul, Korea). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies (Jackson Immunoresearch, West Grove, PA, USA). SP600125 was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest purity commercially available and were used as received.

The samples (50 µg) corresponding to total cell lysates were run on 10-12% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked and incubated with the respective primary antibody to a 1:500 final dilution (PCNA, Bcl-2, pBcl-2 (Ser⁸⁷), Bcl-xL, Bid, Bax, caspase-3, caspase-8, caspase-9, pGSK3β (Ser⁹), GSK3β, and pβ-catenin (Ser⁴⁵), pβ-catenin (Ser^33/37), β-catenin, pJNK (Thr¹⁸³ and Tyr¹⁸⁵), JNK, pERK (Tyr²⁰⁴), ERK, AKT, pAKT (Ser473), MMP2, MMP9, SNAIL, E-cadherin, vimentin fibronectin, cytokeratin, β-actin, and β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, and Abcam) for 24 h at 4 °C. Immunoreactivity was visualized by probing with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and detected using the ECL kit (Santa Cruz Biotechnology).