Figure 5.

The differential effects of Salvador on autophosphorylation of Mst2 in vitro or in cells. A, purified Mst2 was incubated with ATP and Mg²⁺ with or without 100 μm Salvador SARAH. Samples were taken at different times (0, 5, 10, 15, 30, 60, and 120 min), quenched with EDTA, and analyzed by Coomassie-stained SDS-PAGE and the level of autophosphorylation was determined by Western blotting with an antibody that recognized phosphorylated Thr-180 (pT180 Mst2). The assay was run three times and representative blots are shown. B, HEK293T cells were transiently transfected with the indicated plasmids (HA-Mst2 and HA-hSav1). Normalized cell lysates were analyzed by Western blotting for protein expression with antibodies that recognize the epitope tag (HA), the phosphorylated activation loop of Mst2 (pT180 Mst2), or β-Actin as a loading control. Representative blots from one experiment (top) and a scatter plot of 11 replicates (bottom) are shown. Data are plotted as pThr-180 Mst2 intensity divided by total HA-Mst2 intensity (p value ≤ 0.001).