FIG. 4.

In vivo phosphorylation and activation of MK3 by arsenite. Primary WT and MK2^−/− MEF cells were starved overnight and then stimulated with 200 μM arsenite (Ars) for 30 min. The protein lysates were prepared. (A) The lysates were immunoblotted for phosphorylated MK2/MK3 and for total MK2. (B) MK2/MK3 kinase activity against recombinant Hsp25. Endogenous MK2 and MK3 were precipitated indirectly by using catalytically dead mutant GST-p38 (TGY/AGF) coupled to glutathione-Sepharose beads. The beads were used in a kinase reaction with recombinant Hsp25 as a substrate. The reaction mixture was resolved by SDS-PAGE immunoblotted for MK3 (WB), and then Hsp25 phosphorylating activity was detected by phosphorimaging (autoradiogram). (C) In-gel Hsp25 kinase assay of precipitates from GST-p38 (TGY/AGF) pull-down in WT and MK2^−/− MEFs.