Figure 5. PP2Cϵ directly dephosphorylates ASK1 in vitro.

HEK-293 cells were transiently transfected with an expression plasmid encoding ASK1–FLAG. Exogenously expressed ASK–FLAG was then immunoprecipitated (IP) with an anti-FLAG antibody (αFLAG) and incubated with various amounts of recombinant PP2Cϵ (rec. PP2Cϵ; lanes 1 and 2, none; lane 3, 0.1 μg; and lane 4, 0.3 μg) in vitro. Incubation mixtures were immunoblotted (WB) with anti-[phospho-ASK(Thr⁸⁴⁵)] (αpASK1) or anti-FLAG (αFLAG) antibodies. Coomassie Brilliant Blue (C.B.B.) staining of recombinant PP2Cϵ following blotting on to PVDF membranes is also shown (bottom panel). The results are from one of two reproducible experiments.