C86 binding to Hsp40/DnaJ family members likely involves the highly conserved J-domain.
(A) Biotin-C86 or -inactive analog (b-IA) binding to FLAG-DnaJ family members from transfected HEK293 cell lysates (left) or to endogenous Large T antigen from COS7 cell lysate (right) as assessed by streptavidin-bead IP and WB.
(B) Biotin-C86 binding to WT or H/Q mutant V5-DnaJB6b (left) or V5-DnaJB8 (right) as assessed by streptavidin-bead IP and WB.
(C) Biotin-C86 binding profile from 22Rv1 CRPC cells incubated with excess unlabeled vehicle (DMSO) or 50 μM C86 for 1 hour as assessed by streptavidin-bead IP and WB.
(D) 22Rv1 CRPC cells were transfected with FLAG-Hsp70 and treated with vehicle or 10 μM C86 for 1 hour. Lysate was then probed with FLAG-beads and bound proteins were detected by WB.
Experiments were repeated at least three times.