Fig. 3

FBW7 binds to and mediates the proteolytic degradation of YTHDF2 in ovarian cancer. a FBW7 interacts with YTHDF2 by the reciprocal co-IP assays in SKOV3 cancer cells. b The IF staining indicates the co-localization of FBW7 and YTHDF2 in the cytoplasm. c Ectopic expression of FBW7 reduces the YTHDF2 protein levels in SKOV3 and OVCAR429 cells, which can be blocked by the proteasome inhibitor MG132 (20 μM). d The half-life of YTHDF2 is shortened upon FBW7 overexpression. The cells were treated with 100 mg/ml of cycloheximide (CHX) and harvested at different time points as indicated. The quantification of YTHDF2 expression is shown in the lower panel. Overexpression of FBW7 shortened the half-life of YTHDF2 from 7.0 h to 3.3 h. e FBW7 promotes ubiquitination of YTHDF2 in 293 T cells. Cells were transfected with combinations of plasmids encoding YTHDF2, FBW7 or His-Ub as indicated, and treated with MG132 (20 μM) or DMSO for 6 h before harvested for in vivo ubiquitination assay. Bound and input proteins were detected by IB assays with the indicated antibodies. f The ubiquitination of YTHDF2 is mediated by FBW7. Cells were transfected with combinations of plasmids encoding YTHDF2, FBW7 or His-Ub as indicated, and treated with MG132 (20 μM) or DMSO for 6 h before harvested for the IP assays using the anti-YTHDF2 antibody. Results were presented as mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups