Figure 3. USP5 is Required for ERK-Mediated METTL3 Stabilization.
(A) Comparison of METTL3 and WTAP protein levels in mESCs and A375 stable transfectants by immunoblotting (IB).
(B) 293T cells transfected as indicated were treated with MG-132 (10 μM, 8hr) followed by IP/IB.
(C) 293T cells transfected as indicated for 48 hr, followed by cycloheximide (CHX) 10 μg/ml for 0–12 h. Lysates were used for IB to measure the protein levels of METTL3. Density of METTL3 expression was quantified by ImageJ and the relative fold compared to the untreated WT was indicated and plotted at the right panel.
(D) BRAF expression promotes METTL3-USP5 interaction. Lysates of 293T cells transfected as indicated for 48 hr were subjected to IP with anti-myc antibody followed by IB.
(E) USP5 decreases ubiquitination of METTL3. Lysates of 293T cells transfected as indicated for 48 hr were subjected to IP with anti-myc antibody followed by IB.
(F) Knockdown of SPOP, TRIM28, and ANAPC1 attenuated USP5 inhibition-induced degradation of METTL3. A375 cells were transfected with siRNA for 72 hr or treated with 10 μM EOAI3402143 (EOAI) for 8 hr before IB analysis.