FIG. 5.

Rescue of Hsp25 phosphorylation and p38 level in MK2-deficient MEFs by overexpression of MK3. MK2^−/− immortalized MEF cells were transduced with pMMP-IRES-EGFP vector expressing EGFP in parallel with wild-type MK2, a catalytically dead mutant of MK2-K76R, or MK3 and as a negative control with empty vector expressing EGFP alone. Cells were starved overnight and then were left unstimulated (Ars−) or were stimulated for 40 min with 200 μM arsenite (Ars+). (A) In vivo rescue of Hsp25 phosphorylation and p38-alpha protein levels by overexpressed MK3. The lysates were immunoblotted for phosphorylated and total Hsp25 and p38-alpha and for phosphorylated MK2/3. As an expression control the lysates were immunoblotted for MK2, MK3, and GFP. (B) Cell lysates were incubated in vitro with recombinant Hsp25 and [γ-³³P]ATP, and Hsp25 phosphorylating activity was detected by phosphorimaging.