Journal: Nature Communications
Article Title: Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation
Figure Lengend Snippet: 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 K d values are displayed in the inset. Error bars in the K d 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, Hsp90 Y197E , or Hsp90 Y197F 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
Article Snippet: Antibody sources/clone #’s are as follows: GR (glucocorticoid receptor) monoclonal antibody is from Santa Cruz Biotechnology (cat # sc-393232, 1:5000); GFP monoclonal antibody is from Cell Signaling (cat #2956, 1:1000); GFP-trap beads are from Chromotek (GFP-Trap A, cat # gta-20); anti-FLAG monoclonal antibody (clone M2) is from Sigma (cat # F3165, 1:2000); anti-FLAG resin is from Sigma (M2 anti-FLAG antibody-linked resin); Hsc/Hsp70 antibodies are from Santa Cruz (cat # sc-1059, 1:1000, sc-1060, 1:1000); Aha1 antibody is from Rockland (cat # 600-401-974); p23 antibody is from Assay Designs (ADI-SPA-610, 1:1000); HOP antibody is from Cell Signaling (cat # 4464, 1:1000); FKBP59 antibody is from StressMarq (SMC-139, 1:1000); Cdk4 antibody is from Santa Cruz (sc-601, 1:1000); HA antibody is from Roche diagnostics (rat anti-HA, clone 3F10, 1:1000); anti-c-Myc Agarose Affinity Gel antibody from Sigma (cat # A7470); and penta·His Antibody, BSA-free, is from Qiagen (cat # 34660, 1:5000).
Techniques: Binding Assay, Lysis, Immunoprecipitation, SDS Page, Western Blot, Transfection