Figure 2. FBXW7 Promotes SHOC2 Ubiquitylation, Triggered by MAPK-Mediated Phosphorylation on Thr⁵⁰⁷.

(A) 293 cells were transfected with the indicated plasmids, lysed with 6 M guanidine solution, pulled down with Ni-bead, and followed by IB for GST-SHOC2, or direct IB.

(B and C) 293 cells were transfected with WT SHOC 2 (B) or SHOC2 mutants (C), followed by IP purification and in vitro ubiquitylation. The blot was probed with anti-GST Ab.

(D and E) A549 cells (D) or Miapaca-2 and H1299 cells (E) were treated with EGF or serum for indicated time periods, followed by IB for p-SHOC2 (T507), SHOC2, p-ERK, and ERK.

(F and G) H1299 cells were transfected with MEK1 siRNA (siMEK1) in the presence (G) or absence (F) of EGF or treated with MEK1 inhibitor (PD98059, MEKi) for 48 h, followed by IB.

(H) A549 cells were transfected with Flag-FBXW7 for 48 h in the presence or absence of EGF (50 ng/ml) for 5 min, followed by immunoprecipitation and IB with indicated Abs. WCE, whole-cell extract.

(I and J) HCT116 FBXW7^−/− cells were transfected with indicated plasmids (I) or MEK1 siRNA in the presence or absence of MEKi (PD98059) (J), followed by indicated serum treatment. Cells were then lysed with 6 M guanidine solution, pulled down with Ni-bead, and followed by IB. WCE, whole-cell extract.