Cdc37

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).

Sections or cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, blocked with 5% BSA, and then incubated with indicated antibodies against HA (Santa Cruz, California, USA), AHA1 (Abcam, Cambridge, UK), CDC37 (Abcam) or CD68 (CST, MA, USA) overnight at 4 °C. Alexa Fluor® 488- or 594-conjugated fluorescent secondary antibodies (Life Technologies, Thermo Fisher Scientific) were incubated at 37 °C for 1 h, and nuclei were counterstained with DAPI (Santa Cruz). Images were captured using a confocal microscope (Zeiss LSM 410, Oberkochen, Germany).