Figure 3.

Validation of the Hsp70 proteome isolated by the chemical probes. (A–C) SKBr3 cancer cell extracts were incubated overnight with the indicated biotinylated probes (A,C) or the indicated antibodies (B,C). Protein complexes isolated on streptavidin agarose beads (A,C) or protein G agarose beads (B,C) were probed with the indicated antibodies (A,C) or silver stained (B). (D) Cell lysates immunodepleted with either BB70 or a control IgG were subjected to affinity purification with YK5-biotin1/SBs. Isolated proteins were probed with the indicated antibodies. LE and HE, low- and high-exposure, respectively (E) Experiment setup as in panel A for the indicated cancer cell extracts. (F) As in panel E for cell extracts preincubated for 3 h with the indicated concentrations of YK5. (G) Cancer cells were treated for 24 h with the indicated concentrations of vehicle (DMSO) or YK5, and proteins were analyzed by Western blot. These experiments were repeated twice with comparable results.