Fig. 5.

ERK phosphatases are inhibited by Zn²⁺ in vitro. (A) ERK phosphatase assay using ppERK-His and nickel column purification before Western blotting. Zn²⁺ or chelator (TPA) were either added to cells for 30 min before lysis or added directly to HeLa cell lysate. BCI-hydrochloride and λ-PPase controls were added to cell lysate, and ppERK lane indicates the amount of ppERK added to each sample. Blot is representative of four separate experiments. (B) In vitro inhibition studies of MKP-3 by Zn²⁺ through detection of phosphate group released from 4-methylumbelliferyl phosphate. Colors indicate data points from three separate experiments with a variety of Zn²⁺ concentrations. Purple dots use a variety of chelators and counter ions to buffer the Zn²⁺ concentration for more accurate measurement (SI Appendix, Table S3).