Hsp90β

Whole cell extracts were prepared using a lysis buffer containing 10 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 50 mM NaF and 1% Triton X100 supplemented with Complete Protease Inhibitor Cocktail and PhosStop tablets (Roche). After measuring the protein content via Bradford, lysates were subjected to SDS-PAGE and separated proteins transferred to PVDF membranes (Millipore, Massachusetts, USA). For co-IP, protein extracts (IP - 1 mg, input - 100 μg) were incubated with 3 μl STK33 antibody (0.36 μg/μl) and Protein G-Sepharose (GE Healthcare). For western blot analysis 100 μp protein was used. Membranes were blocked with 5% non-fat dry milk in phosphate buffered saline (PBS) containing 0.2% Tween 20 and incubated over night at 4°C with specific antibodies. For subsequent washes 0.2% Tween 20 in PBS was used. The following antibodies were used: STK33 (Abnova, clone 4F7, #H00065975, dilution 1:1000); HIF-1α (BD Transduction Laboratories, #610959, dilution 1:50); cleaved PARP (Cell Signaling, #9505S, dilution 1:700); cleaved caspase 3 (Cell Signaling, #9661, dilution 1:500); HSP90β, (#clone D-19, Santa Cruz Biotechnology, #sc-1057, dilution 1.500); phospho-PKD/PKCμ - Ser744/Ser748 (Cell Signaling, #2054S, dilution 1:1000) and β-actin (Sigma, #A1978, dilution 1:2000).

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.

Specific antibodies to p53, HSP70, HSP60, HSP90β, β-tubulin, p21, GAPDH, and HSP90β siRNAs were purchased from Santa Cruz (Santa Cruz, CA, USA). Polyclonal antibodies to Bax, Bcl-2, cytochrome c, and β-actin were obtained from Millipore (Temecula, CA, USA). Polyclonal antibodies to caspase-3, -7, -9, PARP, CDK2, -4, -6, cyclin D, cyclin E, N-cadherin, E-cadherin, claudin, AKT, and phospho-AKT^Ser473 were purchased from Cell Signaling (Beverly, MA, USA). Pan-caspase inhibitor (Z-VAD-FMK) was obtained from Enzo life science (New York, NY, USA).
The overall structure flowchart is shown in Figure 6 as follows: