FIGURE 7.

Inhibiting the ATP hydrolytic activity of Hsp70 has no effect on the interaction between tTG and Hsp70, or on the protein cross-linking activity of tTG. A, HeLa cells ectopically expressing V5-Hsc70 and Myc-tTG were treated without (Untreated) or with myricetin, methylene blue, or VER 155008, as indicated, and lysed. Immunoprecipitations (IP) with a Myc antibody were performed on the cell extracts, followed by SDS-PAGE and Western blot analysis using V5 and Myc antibodies. Nonspecific mouse IgG control antibody and beads-only control immunoprecipitations were performed on the extracts from the untreated cells to show that the Hsc70-tTG interaction was specific. B, serum-starved cultures of MDAMB231 cells were treated without (Untreated) or with T101, myricetin, methylene blue, or VER 155008 and then lysed. The extracts were immunoblotted with tTG and actin antibodies (top panels) and assayed for transamidation activity as read-out by the incorporation of BPA into lysate proteins (bottom panel).