Cdc37

Western blotting and immunoprecipitations were performed using the following primary homemade antibody: a monoclonal antibody against Morgana (P1PP0) was generated in our laboratory using GST-morgana fusion protein as an antigen^{20 (link)}. Other commercially antibodies were used: Vinculin (Sigma, SAB4200080, 1:5000), CHORDC1 (Sigma, HPA041040, 1:1000), MMP9 (Abcam, ab76003, 1:1000), MMP2 (Santa Cruz, 8835, 1:500), HSP90 (Santa Cruz, 13119, 1:1000), P-IkBα Ser32-36 (Santa Cruz, 8404, 1:500), P-IkBα Tyr42 (Abcam, ab24783, 1:1000), IkBα (Cell Signaling, 4814, 1:1000), IKKα (Cell Signaling, 11930, 1:1000), IKKα (Cell Signaling, 2682, 1:100 for co-immunoprecipitation experiments), IKKβ (Cell Signaling, 8943, 1:1000), IKKγ (Santa Cruz, 8032, 1:500), αTubulin (Sigma, T5168, 1:8000), MBP (Cell Signaling, 2396, 1:1000), TNF-R1 (Santa Cruz, 8436, 1:500), TAK-1 (Santa Cruz, 166562, 1:500), p-AKT (Cell Signaling, 9271, 1:1000), AKT (Cell Signaling, 4691, 1:1000), CDC37 (Santa Cruz, 5617, 1:500), P-IKKα/β (Cell Signaling, 2697, 1:1000).
TNFα (300-01A and 315-01A) was purchased from Peprotech, the IKKβ inhibitor PS1145 (P6624), ROCK inhibitor Y27632 (Y0503), the PTEN inhibitor VO-OHpic (V8639), and the AKT inhibitor GSK69093 (SML0428) were from Sigma. BEZ235 (S1009) was purchased from Selleckchem.

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.

Antibodies and sources: CDK9, pCDK9, RNAPII, phospho-RNAPII, TGM2, Cyclin B1, Cyclin E1, and NUP98 (Cell Signaling Technology); CDC37, SPT5, Stomatin, PLK1, Cyclin A1 (Santa Cruz Biotechnology); BRD4, Cyclin T1 and GFP (Abcam); Caspase-8 (Enzo Life Sciences); β-Actin, pCDK9, Vimentin, and Flag (Sigma-Aldrich).
Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assay (Promega); BrdU kit (Roche); Thymidine, 5-ethynyl uridine (EU), Azide-fluor 488 (Sigma-Aldrich); AnnexinV and 7AAD (BD); [γ-32P] ATP (3000Ci/mmol, Amersham Pharmacia); Trail, FasL (Enzo Life Sciences); PLA assay kit (Olink Biosciences); BAY1251152, Cisplatin, and Carboplatin (Selleckchem); BioCoat Matrigel invasion chamber (Corning); Migration chamber (Ibidi); RNeasy Plus kit (Qiagen), active GST-CDK9/Cyclin K (SRP5012, Sigma-Aldrich).
The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene); p3xFlag-CMV-7.1 (E7533, Sigma); pGEX-5X-3 (28–9545-55, GE Healthcare) and pEGFP-C2 (6083-1, Clontech). All siRNAs and primers were from Sigma-Aldrich.