Because our assay uses recovery of proteins with DNAJB8 H31Q as a proxy for affinity, changes in protein abundance would complicate the interpretation. To avoid perturbation to proteome abundance, we optimized the treatment time to be short enough to preclude transcriptional and translational remodeling of the cell while still inducing protein destabilization. The primary misfolded protein stress response in the cell is the Heat Shock Response (HSR), in which the presence of nuclear or cytosolic misfolded proteins activates the transcription factor HSF1, leading to increased transcription and translation of chaperones and degradation factors and consequent restoration of cellular protein homeostasis . A primary chaperone target of HSF1 is the cytosolic Hsp70 HSPA1A. We validated that 30 min 1 mM of acetochlor in serum-free media followed by a 16 h recovery, induces HSR target HSPA1A in HEK293T cells in cells overexpressing Flag DNAJB8 H31Q (Figure ). Treatment with 1 mM acetochlor for 30 minutes in serum-free media was thus chosen for profiling using our AP-MS strategy.