Supplementary Information
Primer sequences for cloning P1 CCAAGCTAGCATGGTGGACTACAGCGTG P2 TAGCGGCCGCAGGTCACACACTGACATC P3 TGTCCTCGAGTCACTTTTGGCTGTCATC P4 GATCAAGCACTTTGGCAGGCTTCGCCGCTGG (M164R) P5 AGGAGAAATGTGCACGCATGGAGCAGGTGGC (L205R) P6 CAGATCAAGCACTTTGGCGCCCTTCGCCGCTGGGATGAC (M164A) P7 GGAGGAGAAATGTGCAGCCATGGAGCAGGTGGCC (L205A)
Oligos annealed for HA tags H1 TTAAGCTAGCATGTACCCATACGACGTGCCTGACTACGCCA H2 CTAGTGGCGTAGTCAGGCACGTCGTATGGGTACATGCTAGC
Oligos annealed for FLAG tags F1 TTAAGCTAGCATGGATTACAAGGATGACGACGATAAGGCCA F2 CTAGTGGCCTTATCGTCGTCATCCTTGTAATCCATGCTAGC
Tags were added to the N-terminus of CDC37 siRNA sequences CDC37 siRNAs (O3; ACACAAGACCUUCGUGGAA and O4; CGGCAGUUCUUCACUAAGA) and control siRNAs (IC3; ACACAAGAUUCCCGUGGAA and IC4; CGGCAGUUCACUUCUAAGA) were obtained from Dharmacon. siRNA targeting the 3’ UTR of CDC37 (O1; CAGCAAUGAUCUUCCAAUA) and Allstars negative control were obtained from Qiagen, Crawley, UK. HSP90α siRNA (GAACGACAAGUCUGUGAAG), HSP90β (GCACAACGAUGAUGAACAG) and corresponding negative controls HSP90α-IC (GAACGACAGAUCUGUGAAG) and HSP90β-IC (GCACAACGAGUAUGAACAG) were obtained from Dharmacon.
Antibodies for western blotting and immunoprecipitation Antibodies used were as follows: CDC37 sc-5617; CDK4 sc-260; CDK6 sc-177; CRAF Sc- 227; ERBB2 sc-284 and HSC70 sc-7298 obtained from Santa Cruz, CA, USA. AKT #9272; P-AKT #9271, P-ERK1/2 #9101, P-MEK1/2 #9121, MEK1/2 #9122, P-RB S780 #9307 and S795 #9301 were from Cell Signalling. Cyclin D1 MS-210 was obtained from Neomarkers, CA, USA; FLAG F1804 from Sigma-Aldrich, Dorset, UK; GAPDH MAB374 from Chemicon
1 International, CA, USA; HA HA.11 from Covance, CA, USA; HSP90 SPA-830 from Stressgen Bioreagents, Victoria, Canada and Hypophospho-RB 554164 from BD Pharmingen, Belgium. Polyclonal P-CDC37 S13 antibody was raised to residues 9-19 of human CDC37 (48). ERK2 was a gift from Professor Chris Marshal, Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, UK. For FLAG immunoprecipitation, anti-FLAG M2 affinity gel (Sigma) was used according to the manufacturer’s protocol.
Densitometry of western blots: Blots from multiple independent experiments were scanned and analysed using ImageJ 1.47v software. The values obtained were normalised to corresponding GAPDH levels (except in the case of serial dilutions of the lysates or for immunoprecipitations) and expressed as a ratio relative to an appropriate control.
Isothermal titration calorimetry and Kd determinations: Heat of interaction was measured on an ITC200 microcalorimeter (Microcal), with a cell volume of 200l, under the same buffer conditions (20mM Tris pH 7.5 containing 1mM EDTA and 5mM NaCl). Ten aliquots of 3.7l of 325M of either wild type human Cdc37, or M164A or M164R/L205R mutant protein were injected into 30M human Hsp90 at 30oC. Heats of dilution were determined in a separate experiment by diluting protein into buffer, and the corrected data fitted using a nonlinear least square curve-fitting algorithm (Microcal Origin) with three floating variables: stoichiometry, binding constant and change in enthalpy of interaction.
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