Fig. 4.
Assembly of complexes of Cdc37 and Hsp90 phosphomimetic variants with clients and cochaperones. a Binary complex formation between Cdc37 variants and bRaf (left), and between Hsp90β variants and Cdc37 (right), followed by ITC. The corresponding Kd values are displayed in the inset. Error bars in the Kd values correspond to the errors resulted in fitting of the data into a single binding site model. b HEK-293 cells were cotransfected with indicated HA-tagged Cdc37 and FLAG-tagged bRaf plasmids. After cell lysis, proteins were immunoprecipitated with anti-FLAG resin for 1 h at 4 °C with rotation. Bead pellets were washed and analyzed for Cdc37 interaction by SDS-PAGE/western blot, using anti-HA antibody. c HEK-293 cells were transfected with FLAG-tagged Hsp90, Hsp90Y197E, or Hsp90Y197F plasmids. After cell lysis, proteins were immunoprecipitated with anti-FLAG resin for 1 h at 4 °C with rotation. Bead pellets were washed three times before analysis by SDS-PAGE/western blot. Co-precipitating endogenous Hsp70, Aha1, p23, Hop, Fkbp59, and Cdk4 were detected with specific antibodies. d HEK-293 cells were transfected with the indicated Hsp90, androgen receptor (AR), and glucocorticoid receptor (GR) plasmids. Proteins were precipitated with GFP-Trap resin (left) or ANTI-FLAG M2 agarose (right) for 1 h at 4 °C with rotation. Bead pellets were washed three times with lysis buffer before analysis by SDS-PAGE/western blot as indicated. AR was visualized with anti-GFP antibody, GR was visualized with a specific antibody, and Hsp90 was visualized with anti-FLAG antibody