Raf 1

DU145 cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris–HCl [pH 7.5], 2 mM ethylenediaminetetraacetic acid) and 15 μg of protein was separated on 6–12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Proteins were transferred to polyvinylidene difluoride membranes and then membranes were blocked in PBS with 0.1% Tween-20 containing 1% BSA and 1.5% skim milk for 1 h. After washing, the membranes were probed with primary antibody at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blot was developed using the EZ-western detection kit (Daeillab Service, Co., Seoul, Korea). Anti-cleaved caspase-8, −cleaved caspase-3, −PARP, −JNK, −p38, −p-ERK1/2, −p-SRC (Tyr-416 and Tyr-527), −SRC, −p-STAT3, −STAT3, −p-PI3K, −PI3K, −p-AKT (Ser-473), −AKT, −Ras, −p-Raf1 (Ser-259 and Ser-338), −p-MEK1/2, −p-p90RSK (Ser-380), and -RSK1/RSK2/RSK3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-Actin, −p-JNK, −p-p38, −ERK2, and -Raf1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz). The band intensities of specific antibodies were normalized and analyzed by ImageJ (Broken Symmetry Software, version 1.4.3.67).

Cells and tissue samples were collected and incubated in the RIPA buffer (Sigma-Aldrich) to obtain proteins. Approximately 30 μg of total proteins were loaded into each lane of 10-15% SDS-PAGE, separated in electrophoresis at a constant pressure of 160 V for about 1-2 h, and transferred onto nitrocellulose membranes (Millipore). After blocking, the membranes were then incubated with primary antibodies against EGFR (Santa Cruz Biotechnology), phosph-EGFR (Santa Cruz Biotechnology), HER2 (Santa Cruz Biotechnology), RAS (Santa Cruz Biotechnology), RAF1 (Santa Cruz Biotechnology), MEK1/2 (Cell Signaling), phosph-MEK1/2 (Cell Signaling), ERK1/2 (Cell Signaling), phosph-ERK1/2 (Cell Signaling), and β-actin (Santa Cruz Biotechnology). β-actin was loaded as an internal reference. Bands were then treated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). Bands were developed using chemiluminescence substance (Thermo Scientific). All experiments were conducted three times independently with similar results. A representative result for Western blots was provided.

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/streptomycin (P/S) for cell culture, blocking solution for Western blot, MANT-ADP for ATPase binding assay, Opti-MEM medium, siRNA (HSP90β, scramble), lipofectamin 2000, lipofectamine RNAi Max for transfection, qRT-PCR kit were obtained from Life Technology. RIPA lysis buffer, ECL were purchased from Thermo; protease inhibitors and phosphor-stop were acquired from Roche. Strep-tactin superflow plus were purchased from Qiagen. Bradford protein assay kit was purchased from Bio-rad. High Throughput Colorimetric ATPase Assays kit were from Innova Biosciences. Caspase-3 activity assay kit was purchased from Cell Signaling. Apoptosis detection kit was purchased from BD. For antibodies, Raf-1, IKK-1/2, HSP90β, p-HSP90β, HSP70, CDC37, PPIA, p-MEK, MEK, p-ERK, ERK, GAPDH, PARP and Ub antibodies were purchased from Santa Cruz; Anti-flag antibodies was from Sigma; CDK4, p-Rb, Caspase-9, eEF2 and Strep-HRP antibodies were from Cell Signaling. All chemicals not listed above were purchased from Sigma-Aldrich.

Xenografts were formalin-fixed at least for 24 h and paraffin-embedded. Sections were evaluated by hematoxylin & eosin (H&E) staining. Immunohistochemistry (IHC) was performed on additional sections for HER2 (Dako), Phospho-Akt (Ser473) (Cell signalling), FGFR2 (Abcam), and Raf-1 (Santa Cruz) following the manufacturer's instructions. Western blotting of phosphorylated HER2 (p.Y1248) was performed using antibody cat Nr-06-229 (Millipore).

Primary antibodies used for western blotting include Flag M2 at 1:3000 (Sigma, F3165, St. Louis, MO), Flag-HRP at 1:1000 (Sigma, A8592, St. Louis, MO), GST Z-5 at 1:3000 (Santa Cruz, sc-459, Dallas, TX), rabbit GST-HRP at 1:1000 (Sigma, A7340, St. Louis, MO), Aurora kinase A at 1:500 (Cell Signaling, 4718, Boston, MA), rabbit pERK and ERK (Cell Signaling, 4370, 9102, respectively, Boston, MA), pMEK and MEK (Cell Signaling, 9154, 4694, respectively, Boston, MA), pRaf-1 (Cell Signaling, 9427, Boston, MA) and Raf-1 (Santa Cruz, sc-133, Dallas, TX) at 1:1000, and Ras (BD Transduction Laboratories, 610002, San Jose, CA) at 1:1000. Secondary antibodies include goat anti-rabbit IgG (Santa Cruz, sc-2004, Dallas, TX) and goat anti-mouse IgG (Santa Cruz, sc-2005, Dallas, TX) and were used at either 1:2500 or 1:5000 dilutions. Antibodies used for immunoprecipitation include Aurora A (Sigma, A1231, St. Louis, MO), Ras (ThermoScientific, MA1-012X, Waltham, MA), and IgG (Santa Cruz, sc-2027, Dallas, TX) at 1:50 dilution.

Antibodies used: GAPDH (HyTest Ltd., clone 6C5, cat. # 5G4), caspase-3 (Abcam, ab4051), Cdk6 (Abcam, ab3126), Ras (Stressgen, now Enzo Life Sciences; ADI-KAP-GP001), p-ERK (Santa Cruz, sc-7383), Hsp70 (Abcam, ab181606), p27Kip1 (Cell Signaling, #2552), Nr2f1 (ABGENT, #AP14218a), tubulin (Merck, CP06), Cdk4 (NeoMarkers, MS-616-P1), Raf-1 (Santa Cruz Biotechnology, sc-133), Akt (Cell Signaling, #9272). Transfection reagents: jetPRIME (Polyplus, #114–15), polyethylenimine (PEI) (Polysciences, # 621405). Hsp90 inhibitors: geldanamycin (LC Laboratories, G-4500), PU-H71 (Tocris, #3104), pochoxime A (kindly provided by Nicolas Wissinger, University of Geneva). Other inhibitors: AZ628 (Tocris, #4836), PD98059 (Tocris, #1213), cycloheximide (Sigma, C7698), rapamycin (LC Laboratories, R-5000), aphidicolin (ACROS, BP615-1), hydroxyurea (fluorochem, #043351). All compounds were dissolved in DMSO. Growth factors: insulin (Sigma, I6634), recombinant human epidermal growth factor (EGF) (Lonza, CC-4017). Metabolic assay reagents: Oligomycin (SIGMA, 7535), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (SIGMA, C2920) and rotenone (SIGMA, R8875) with antimycin (SIGMA, A8674).