Fig 2: Knockdown of CRAF and MEK1/2 can abolish the efficiency of erianin. a The expression of CRAF, MEK1, and MEK2 in A375, SK-MEL-2, and HCT116 cells expressing shRNA-mock or shRNA of both CRAF and MEK1/2 was evaluated by western blotting. b MTT assay was evaluated in A375, SK-MEL-2, and HCT116 cells after dual knockdown of CRAF and MEK1/2. The asterisks (*p < 0.05; **p < 0.01; ***p < 0.001) indicate a significant difference between shRNA-mock and shRNA-CRAF/MEK1/2 cells. c Anchorage-independent growth was assessed in A375, SK-MEL-2, and HCT116 cells expressing shRNA-mock or shRNA-CRAF/MEK1/2. Positive colony areas in anchorage-independent growth were calculated using Image-Pro Plus software. Scale bars: 400 μm. Data were shown as means ± S.D. The asterisks (*p < 0.05; **p < 0.01) indicate a significant difference. d Expression of CRAF and MEK1/2 in melanoma and colorectal cancer cell lines. e Western blotting of CRAF or MEK1/2 after knocking down CRAF or MEK1/2 in A375 and SK-MEL-2 cell lines. f Western blotting of Flag-CRAF or Flag-MEK1/2 after transfected with mutant CRAF or MEK1/2 in A375 and SK-MEL-2 cell lines described in (e). g Rescue assay (MTT) after knocking down MEK1/2 or CRAF followed by overexpression of MEK1/2 WT/2 A or CRAF WT/K375A, respectively, in A375 and SK-MEL-2 cell lines. h Rescue assay (anchorage-independent growth assay) after knocking down MEK1/2 or CRAF followed by overexpression of MEK1/2 WT/2 A or CRAF WT/K375A respectively in A375 and SK-MEL-2 cell lines. Data were shown as means ± S.D. Scale bars: 400 μm. The asterisks (*p < 0.05; **p < 0.01) indicate a significant difference

Fig 3: Impaired phosphorylation enhanced poly-ubiquitination of TET2. a IB analyses of TET2 IP products and WCL derived from HCC827 cells. IgG was used as a negative control. b HCC827 and its derivative cells with knockout of either MEK1 or MEK2 were subjected to IB analysis. c Relative TET2 mRNA level in indicated cells as described in (b). d IB analyses of HCC827WT cells or HCC827MEK1-KO cells reintroduced with empty vector, MEK1WT or MEK1SA. e CRISPR-Cas9 mediated MEK1 knockout of 293T cells were transfected with indicated constructs and treated with MG132 (10 μM, 12 h) before IP and IB analyses. f IB analyses of HCC827-OR cells without or with the overexpression of constitutive MEK1 (MEK1CA). g Flow chart to show Flag IP products from 293TMEK1-KO cells transfected with indicated constructs and treated with MG132 were subjected to phosphoproteomics. h Alignment of putative TET2 phosphorylation residues among different species. The asterisks below indicate the four potential phosphorylated amino acids. i IB analyses of Flag IP products from 293TMEK1-KO cells treated with MG132 (10 μM, 12 h) as shown in (g). pSer pan phospho-serine, pThr pan phospho-threonine, pTyr pan phospho-tyrosine. j IB analyses of endogenous TET2 immunoprecipitated from PC-9 (up) or HCC827 (bottom) cells with or without osimertinib treatment. k 293TMEK1-KO cells were transfected with indicated constructs and treated with MG132 (10 μM, 12 h) before harvested for Flag immunoprecipitation, the IP products and WCL were subjected to IB analyses. l 293TTET2-KO cells were transfected with indicated constructs and treated with MG132 (10 μM, 12 h) before harvested for Flag immunoprecipitation, the IP products and WCL were subjected to IB analyses. All data are representative of at least two independent experiments

Fig 4: MEK1 is cleaved in a caspase‐dependent manner during apoptosis. (A) U2OS and A375 cells were treated with etoposide (60 μm for 24 h) and the expression level of endogenous MEK1 was analyzed by immunoblotting using an antibody against the N‐terminal region (amino‐acid residues 2–18) of MEK1. Etoposide induced the appearance of the cleaved 35‐kDa form of MEK1 (Cleaved). PARP, BRaf, MEK2, and ERK1/2 in cell extracts were also probed with the appropriate antibodies. Actin served as a loading control. Short, short exposure. Long, long exposure. FL, full‐length. *, nonspecific bands. (B) U2OS cells were stimulated with etoposide (60 μm) for the indicated times. The cleavage of MEK1 and PARP was analyzed by immunoblotting. (C, D) A549 (C) and Jurkat (D) cells were exposed to osmotic stress (0.6 m sorbitol) or anti‐Fas antibody (200 ng·mL−1) for the indicated times. Immunoblotting was performed as in (A). (E, F) U2OS (E) and Jurkat (F) cells were stimulated with etoposide (60 μm for 24 h), osmotic stress (0.6 m sorbitol for 6 h), or anti‐Fas antibody (200 ng·mL−1 for 8 h) as indicated. The induced proteolytic cleavage of PARP and MEK1 was analyzed by immunoblotting. (G) U2OS and Jurkat cells were stimulated with etoposide (60 μm for 24 h) or anti‐Fas antibody (200 ng·mL−1 for 10 h) as indicated. The cleavage of MEK1, caspase‐9, caspase‐8, and caspase‐3 was analyzed by immunoblotting using appropriate antibodies. In (A, E–G), cells were pretreated with (+) or without (−) a pan‐caspase inhibitor Z‐VAD‐FMK (100 μm), a caspase‐8 inhibitor Z‐IETD‐FMK (20 μm), or a caspase‐9 inhibitor Z‐LEHD‐FMK (20 μm) for 2 h before stimulation as indicated.

Fig 5: The MEK2-E2 interaction occurs during the late stage of CSFV replication and requires the C termini of MEK2 and E2. (A) Co-IP analysis of E2 and MEK2 in CSFV-infected cells. PK-15 cells were infected with CSFV, and the cells lysates were collected at 24, 48, and 60 h postinfection (hpi) and precleared with protein G-agarose, followed by incubation with a rabbit anti-phosphorylated MEK2 (anti-p-MEK2) or a rabbit anti-MEK2 monoclonal antibody (MAb) (1:500) at 4°C for 6 h and incubation with protein G-agarose at 4°C for 6 h. The bound proteins in the agarose were examined by Western blotting using anti-E2 MAb WH303 (1:200). (B) Schematic representation of the porcine MEK2 protein domains and individual MEK2 deletion mutants. (C) GST pulldown analysis of the interaction of GST-tagged MEK2 or its mutants with Flag-tagged E2 expressed in HEK293T cells. The recombinant protein of GST-MEK2 or GST-tagged MEK2 mutants was incubated with glutathione-Sepharose 4B resin (catalog no. 17-0756-01; GE Healthcare). The resin was washed with PBS and incubated with the Flag-tagged E2 protein expressed in HEK293T cells. Immunoblotting analysis was conducted to detect the bound proteins. (D) Schematic representation of the truncated E2 mutants. (E) GST pulldown analysis of GST-tagged MEK2 and Flag-tagged, truncated E2 mutants. The recombinant protein GST-MEK2 was incubated with glutathione-Sepharose 4B resin and then with a series of Flag-tagged, truncated E2 mutants. The bound proteins in the resin were then analyzed by Western blotting as described above.