Hsp27

Plasmids expressing Flag (F)‐Hsp90, K292Q, and K292R mutant were described previously.6The following reagents and antibodies were used: Cdc37, cyclin‐dependent kinase 4 (CDK4), Cyclin D1, Hsp27, p‐(S326)‐Heat shock factor 1, and Serine/threonine‐protein kinase 33 (Abcam, Cambridge, MA, USA); p21^Cip1 (BD Biosciences, Sparks, MD, USA); AKT, p‐AKT, caspase‐3, cleaved caspase‐3, caspase‐8, active caspase‐8, caspase‐9, cleaved caspase‐9, p‐Cdc37, CDK2, CDK6, Cyclin D1, Eukaryotic elongation factor 2 kinase, p‐eEF2K (S366), Hsp70‐Hsp90 organizing protein 1, HSF1, MEK1/2, p‐MEK1/2, Erk1/2, p‐Erk1/2, and poly(ADP‐ribose) polymerase (PARP)1 (Cell Signaling Technology, Danvers, MA, USA); Hsp40/Hdj1, Hsp70, Hsp90α, and p23 (Enzo Life Sciences, New York, NY, USA); Platelet‐derived growth factor receptor β (Merck Millipore, Temecula, CA, USA); Raf‐1 (C20) and Activator of Hsp90 ATPase protein 1 (Santa Cruz Biotechnology, Dallas, TX, USA); LBH589, Sim, mevastatin (Mev), pravastatin, anti‐Myc beads (Selleck Chemicals, Houston, TX, USA); mevalonate, anti‐Flag, β‐actin, anti‐M2, and anti‐HA beads (Sigma‐Aldrich, St. Louis, MO, USA); and Transforming growth factor‐β receptor II (Thermo Fisher Scientific, Grand Island, NY, USA). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA).

Purified LMTK3cat was labeled with an NHS-647 red dye (Nanotemper Technologies) following the manufacturer’s protocol. Purified CDC37 or HSP27 (Abcam, no. ab48740) were serially diluted from 10 μM in MST buffer (Nanotemper Technologies) supplemented with 0.05% Tween 20 before addition of labeled LMTK3 (50 nM). Samples were centrifuged at 12,000g for 5 min before loading into premium coated capillaries. Where specified, C28 (20 μM, 0.4% DMSO) was added to LMTK3 before mixing with CDC37. MST measurements were taken with a Monolith NT.115 system using MO control software at 21°C using 20% excitation power and 60% MST power. Data were analyzed using MO affinity software (Nanotemper Technologies).

For the characterization of 22Rv1, LNCaP, DU145, PC3, and C4-2 cells, 20x10⁵ cells from each cell line were seeded into 100-mm dishes in RPMI1640 medium supplemented with 10% FBS and incubated for 48 h.
For the immunoblotting of 22Rv1 lysates after AMACR siRNA transfection, 22Rv1 cells (20 × 10⁵ per well) were seeded in a 100-mm dish and maintained in RPMI1640 medium supplemented with 10% FBS or CSS without antibiotics overnight (Day 0). The cells were harvested at Day 3 for the AMACR siRNA transfection protocol alone and at Day 4 for the combination therapy of AMACR siRNA transfection and docetaxel treatment (10 nM). The cells were collected and lysed using the RIPA buffer (25 mM Tris, 0.1 M NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, pH 7.4). Expression changes at the protein level were determined by Western blotting. 40 μg of the total protein from each sample was loaded on NuPAGETM 4%-12% Bis-Tris Protein Gels (Invitrogen). The primary antibodies were as follows: ARV7 (1:1000; RevMAb Biosciences, San Francisco, CA, USA), AMACR (1:500; OriGene Technologies, Inc., Rockville, MD, USA). AR (1:1000), HSP27 (heat-shock protein 27) (1:1000), and β-actin (1:1000) were purchased from Abcam in Cambridge, UK. Images were captured and analyzed using a LuminoGraph I (ATTO, Tokyo, Japan).