FIGURE 3.

FBXO31 binds with phosphorylated MKK6. A, serum-starved HEK293 cells were treated with or without UV irradiation and then cultured for 3 h. Lysates from cells treated with UV irradiation were subjected to λ phosphatase treatment in the absence or presence of sodium orthovanadate (Na₃VO₄) at 30 °C for 30 min. Then, cell lysates were incubated with GST-FBXO31-bound beads at 4 °C overnight. Endogenous MKK6 binding was analyzed by immunoblotting (IB). B, HEK293 cells were transfected with myc-FBXO31 plasmids and plasmids encoding an empty vector (EV) or FLAG-tagged MKK6 derivatives (WT, S207E/T211E, S207A, and T211A). Whole cell lysates were immunoprecipitated (IP) with myc antibody, and then FBXO31 and FLAG antibodies were used for immunoblotting.