Pias3

After transfection for 24 hours, cells were lyzed in lysis buffer (Beyotime, Shanghai, China) with the protease inhibitor (Roche, Mannheim, Germany). The protein concentration was examined with a BCA Protein Assay kit (Pierce, Rockford, IL). For sodium dodecyl sulphate polyacrylamide (SDS) gel electrophoresis, equal quantities of total protein (30 μg) were separated on 10% SDS‐PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking in nonfat milk at room temperature for 1 hour, the membranes were incubated with the specific antibodies against PIAS3 (Abcam, Cambridge, MA), STAT3 (Cell Signalling Technology, Beverly, MA), p‐STAT3 (Tyr705) (Cell Signalling Technology) and β‐actin (Cell Signalling Technology) at 4°C overnight. After washing with Tris‐buffered saline‐tween (TBS‐T), the membranes were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody at room temperature for 1‐2 hours. Protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). The Western blots were quantified using Gel‐Pro analyzer software (v4.5, Media Cybernetics, Rockville, Maryland, USA).

Luteolin is purified and provided from Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine (Beijing, China), and the purity of the product was over 98%, detected by HPLC (UV). Gastric tumor cell lines of SGC7901, SGC7901/DDP, HGC27, MGC803, BGC803 and BGC823 were purchased from Keygene (Jiangsu, China). Monoclonal antibodies against HSP-90, STAT3 and phosphor-STAT3 (Tyr705), STAT1 and phosphor-STAT1 (Tyr701), Akt, phosphor-Akt (Ser473), Erk and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) were purchased from Cell Signaling Technology (Boston, MA, USA). Monoclonal antibodies against SHP-1, SHP-2, Bcl-xl, Survivin, Mcl-1, SOCS3, PIAS3, gp130, ki0–67, activated caspase-3 and GAPDH were purchased from Abcam (Burlingame, CA, USA). BCA Protein Assay Kit was purchased from Pierce (Rockford, IL, USA). l-Ascorbic acid was purchased from Cayman (Ann Arbor, MI, USA), α-Tocopherol was purchased from Selleckchem (Houston, TX, USA). DCFDA was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Antibodies against iNOS, CD206, Ubc9, PIAS3, and F4/80 were purchased from Abcam (Abcam, Cambridge, UK); IRF1, IRF2, IκBα, phosphorylated p65, phosphorylated signal transducers and activators of transcription 1 (STAT1), and SUMO-1 were from Cell Signaling Technology (Danvers, MA, USA); p65, p50, p52, PCNA, Stat1 p84/p91, and GAPDH were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); Flag, and c-Myc were from Sigma (Sigma-Aldrich Corp., St. Louis, MO, USA); CD68 was from BMA Biomedicals (BMA Biomedicals, Augst, Switzerland); CD45-PE and CD14-PC5 were from Beckman Coulter (Brea, CA, USA). Anti-mouse or anti-rabbit IgG peroxidase-conjugated secondary antibodies, Alexa Fluor 488-conjugated anti-mouse IgG, and Alexa 568-conjugated anti-rabbit IgG secondary antibodies were purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). Protein A/G was from Wanta Cruz Biotechnology. 5-Azacytidine (5AZ), lipopolysaccharide (LPS), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and MG132 were purchased from Sigma (Sigma-Aldrich Corp.).