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. 2020 Apr 27;10(13):5671–5686. doi: 10.7150/thno.42539

Figure 1.

Figure 1

Mitogen stimulates Mettl3 conjugated to SUMO-1. (A) Mettl3 was overexpressed in MHCC97H cells by transfection with Flag-tagged wild type Mettl3. Subsequently, the cells were transfected with His-SUMO1, -SUMO2, -SUMO3, and HA-Ubc9 or siUBC9. Immunoprecipitation (IP) and western blotting analyses were performed with the indicated antibodies for the SUMOylation assay. (B) Confocal immunofluorescence of endogenous Mettl3 and SUMO1 proteins in MHCC97H cells (Mettl3, green; SUMO1, red). Both Mettl3 and SUMO1 were observed in the nucleus and cytoplasm. Scale bars, 50 μm. (C-F) Representative IP immunoblot analysis was conducted with the anti-Mettl3 or anti-SUMO1 antibody and whole-cell extracts from HepG2 or MHCC97H cells incubated in serum-containing or serum-free medium for 24 h. (C and E) IP analysis was performed with anti-Mettl3 antibodies in HepG2 and MHCC97H cells. (D and F) IP analysis was performed with anti-SUMO1 antibodies in HepG2 and MHCC97H cells. (G) IP immunoblotting analysis examining SUMOylation of endogenous Mettl3 from whole-cell extracts after the addition of 5%, 10%, and 20% serum with anti-Mettl3 antibody or normal IgG, followed by western blotting with the indicated antibodies. (H) IP immunoblotting analysis examining SUMOylation of endogenous Mettl3 from whole-cell extracts after the addition of low (10 ng/ml) or high (20 ng/ml) concentration HGF with anti-Mettl3 antibody or normal IgG, followed by western blotting with the indicated antibodies.