Ciap2

Total protein was isolated and subjected to western blotting analysis as described previously. The following antibodies were used for the western blottings: rabbit polyclonal antibodies detecting XIAP, cIAP1, cIAP2, survivin, cyclin D1, CDK4, Akt, p44/p42 MAPK (Erk) and c-jun (Santa Cruz Biotechnology, Santa Cruz, CA, USA); EGFR, cyclin D3, phospho-serine473 Akt, phospho-p44/p42 MAPK (Erk), cleaved PARP (p85), caspase-3, phospho-p38, p38 and phospho-c-jun (Cell Signaling Technology Inc., Danvers, MA, USA); and β-actin (Sigma Chemical Co., St. Louis, MO, USA). After incubation with the corresponding secondary antibodies for 1 h, signals were detected using an Enhanced ChemiLuminescence kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) followed by autoradiography.

The ANKRD49 rabbit polyclonal antibody was purchased from Abcam (U.K). The Flag mouse monoclonal, Beclin 1, LC3A/B, p65, p62 and GAPDH antibodies were purchased from Cell Signaling Technology (USA); the β-actin and cIAP2 antibodies were purchased from Santa Cruz Biotechnology (USA). HRP-conjugated secondary antibodies were obtained from Zhongshanjinqiao Company (China). Alexa Fluor 488-conjugated goat anti-rabbit antibody, Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 546-conjugated goat anti-rabbit antibody were purchased from Life Technologies (USA).

Based on Lee et al. [25 (link)], Western blotting was conducted in A549/CR and H460/CR cells exposed to PGG (0, 6.25, 12.5 and 25 μM) for 48 h. In brief, whole cell lysates were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail, and isolated proteins in the supernatants were quantified and electrotransferred onto a Hybond enhanced chemiluminescence (ECL) transfer membrane. The membranes were probed with primary antibodies for PTEN, p-AKT, PARP, cellular inhibitor of apoptosis 1 (c-IAP1), c-IAP2, Survivin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Caspase-8, -9, -7, Cleaved caspase-3, BAX, Bcl-2, Bcl-xL, p-ATR, p-Chk2, p-BRCA-1, p-H₂AX (Cell signaling Technology, Danvers, MA, USA), p53 (Oncogene Research Products, San Diego, CA, USA), XIAP (Becton Dickinson and Company BD Biosciences, San Jose, CA, USA), β-actin (Sigma Aldrich, St Louis, MO) and horseradish peroxidase-conjugated secondary antibody. Additionally, nuclear extract was isolated for DNA damage proteins using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Scientific, Rochester, NY, USA).

The MKN28 human gastric carcinoma cells obtained from the American type culture collection (Manassas, VA, USA) were cultured in RPMI medium (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 1 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Antibodies against TRAIL, DR4, DR5, Fas, FasL, XIAP, a cellular inhibitor of apoptosis protein-1 (cIAP-1), cIAP-2, caspase-3, -8, -9, Bcl-2, Bax, Flip_L, Flip_S, PARP, β-catenin, and PLCγ1 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). An antibody against β-actin was from Sigma (Beverly, MA, USA). Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham (Arlington Heights, IL, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were obtained from Calbiochem (San Diego, CA, USA). All other chemicals not specifically cited here were purchased from Sigma–Aldrich (St. Louis, MO, USA). All these solutions were stored at −20 °C. Stock solutions of 4,6-diamidino-2-phenylindole (DAPI, 100 μg/mL) and propidium iodide (PI, 1 mg/mL) were prepared in phosphate-buffered saline (PBS).