Protein Interactome Profiling Using Affinity Purification

Whole-cell protein lysates were generated and analyzed by western blotting as described before [18 (link)]. Whole-cell protein extracts for HSP90 pull-down with geldanamycin were prepared with lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton-X100, protease and phosphatase inhibitors. Lysates (250 μg) were incubated with 40 μg geldanamycin-biotin (Sigma-Aldrich) and 125 μl settled NeutrAvidin Agarose Resin (Thermo Scientific) at 4°C. Immobilized proteins were washed three times, resuspended in Laemmli buffer and subjected to SDS-PAGE and western blotting. The HSP90 pull-down with ATP beads was performed using the Pierce Kinase Enrichment Kit with ATP Probe (Thermo Scientific) according to the manufacturer's instructions. The following antibodies were used: anti-β-actin (Sigma-Aldrich), anti-RAF1 (#9422, Cell Signaling), anti-AKT1 (2H10, Cell Signaling), anti-STK33 (4F7, Abnova), anti-HSP90β (D-19, Santa Cruz), anti-HSP90α (AB3466, Millipore) and anti-p-glycoprotein (anti-ABCB1) (C219, Millipore).