C raf

HM, NM, pCMV‐Vector and pCMV‐FAM83D cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (20 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGTA, 1% NP‐40, 1% sodium, deoxycholate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β‐glycerophosphate, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin and 1 mmol/L PMSF). Total proteins (20‐30 µg) were separated through polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a semi‐dry transfer system (Bio‐Rad). The membrane was incubated at 4°C overnight with specific primary antibodies for FAM83D (Biorbyt, orb183501), AKT (Cell Signal Technology, CST, MA, cat#: 4691), p‐AKT (CST, MA, cat#:4060), ERK1/2 (CST, MA, cat#:9102), p‐ERK1/2 (CST, MA, cat#: 9101), c‐Raf (CST, MA, cat#: 9422), p‐c‐Raf (CST, MA, cat#: 9421), EGFR (CST, MA, cat#: 4267), p‐EGFR (CST, MA, cat#:2234), P38 (CST, MA, cat#:9212), p‐P38(CST, MA, cat#:9211), N‐cadherin (CST, MA, cat#:13116), ZO1 (CST, MA, cat#:8193) and beta‐actin (CST, MA, cat#: 4970) used at a dilution of 1:1000. Subsequently, the membrane was incubated with secondary antibody (Bio‐Rad, CA, cat#: 1706515 in 1:5000 dilution) for 1 hour at room temperature. Clarity™ Western ECL Substrate (Bio‐Rad, cat#: 1705060) was used for the detection of protein signals. The signals were captured using the ChemiDoc™ XRS + Imaging Systems (Bio‐Rad, CA).

Liver tissues were homogenized with T-PER Tissue Protein Extraction Reagent (Thermo Fischer Scientific Inc.) and a protease inhibitor cocktail (Roche Diagnostic, Mannheim, Germany) on ice. Protein concentrations were determined by the Bradford method using a protein assay kit (Bio-Rad laboratories, Hercules, CA). Samples of 50 μg were mixed with SDS sample buffer, heated at 100°C for 10 min and then subjected to SDS-PAGE. The proteins were separated in 12% acrylamide gels and then transferred onto Hybond-ECL membranes (GE Healthcare UK Ltd., Buckinghamshire, UK). The antibodies used were against phosphorylated c-Raf (Ser338) (p-c-Raf), Erk 1/2, pErk, p38Mapk, phosphorylated p38Mapk (Thr180/Tyr182) (p-p38Mapk), pElk1 (Ser383), Sek1/Mkk4, phosphorylated Sek1/Mkk4 (Ser80) (pSek1/Mkk4), Sapk/Jnk, phosphorylated Sapk/Jnk (Thr183/Tyr185) (pSapk/Jnk), cyclin D1, Dusp1 (Cell Signaling Technology), c-Raf (BD bioscience, Franklin Lakes, NJ), and Cyp2e1 (Enzo Biochem Inc.). Equal protein loading was ascertained by western blotting with a β-actin antibody (Sigma-Aldrich corp.).

ISJ and its intermediate, compound 7, were synthesized in our laboratory. GF 109203X (GFX) and Go 6976 (GO) were purchased from Tocris Bioscience (Ellisville, MO). 5-Fluorouracil (5-Fu) was obtained from Sigma (St. Louis, Mo). Primary antibodies were purchased from Cell Signaling Technology (Beverly, MA), including those specific for β-actin (#4970), MMP-9 (#2270), E-Cadherin (#3195), FGFR-1 (#9740), PKCε (#2683), c-Raf (#9422), MEK1/2 (#9122), ERK1/2 (#9102), Phospho-c-Raf (#9427), Phospho-MEK1/2 (#9154), and Phospho-ERK1/2 (#4370). The PKC Isoform Antibody Sampler Kit (#9960) and the Apoptosis Antibody Sampler Kit (#9915) were also purchased from Cell Signaling Technology. Antibodies to p53 (AP062) and p21 (AP021) were purchased from Beyotime (Shanghai, China).

H460, A549, H358, H2122, BEAS-2B, NIH3T3, CCD19-Lu cells were obtained from the American Type Culture Collection and cultured in an environment of 5% CO₂ at 37°C in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), CCD19-Lu cells were grown in MEM medium supplemented with 10% FBS, BEAS-2B cells were maintained in BEBM containing 0.01 mg/ml fibronectin, 0.03 mg/ml bovine collagen type I and 0.01 mg/ml bovine serum albumin. All cells were added 100 units/ml penicillin, and 100 μg/ml streptomycin. Honokiol was purchased from Selleck Chemicals. Antibodies to GAPDH, C-RAF, p-C-RAF, p-AKT (Ser473), p-ERK (Thr202/Thy204), P21, P27, Cyclin D1, Sirt3, Hif-1α, and ERK were purchased from cell signaling technology. Anti-AKT, KRAS antibody were acquired from Santa Cruz Biotechnology.

Western blot analyses were performed as previously described [17 (link), 26 (link), 39 (link)]. Briefly, 90 % confluent NIH3T3 cells were serum-starved for 22 h. Cells were counted and lysed in 1× SDS running buffer, heated at 100 °C for 10 min, and centrifuged to remove debris. Lysates were resolved on 10 % polyacrylamide gels, transferred to nitrocellulose membranes, and blotted with the following primary antibodies: anti-KRAS4A specific antibody (1:500; Santa Cruz Biotechnology, sc-522), anti-RAS antibody (1:2000; Cell Signaling Technology, #3965), anti-actin (1:5000; Sigma-Aldrich, A1978), anti-GFP (1:2000; Cell Signaling Technology, #2555), and phospho-c-RAF, c-RAF, phoshpo-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-AKT, AKT, phospho-S6RP, and S6RP (All 1:2000; Cell Signaling Technology). HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG was used as a secondary antibody.

SDS-PAGE and immunoblotting were performed according to a standard procedure. ECM1, HER3, phosphorylated ERK (p-ERK), ERK, actin, MUC1 and galectin-3 antibodies were obtained from Santa Cruz Biotechnology. c-Raf, p-c-Raf, MEK, p-MEK, Akt, p-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Epidermal growth factor receptor (EGFR) antibody was obtained from Abcam (Cambridge, UK). HER2 antibody was obtained from Thermo Scientific (Waltham, MA, USA).