Fig. 2: Phosphorylation of EZH2 at T367 by DCAF1.
a The indicated recombinant EZH2 proteins were incubated with DCAF1 in the presence of [γ-32P] ATP for 30 min. The reactions were then resolved on 5–20% SDS-PAGE and analyzed by autoradiography (upper panel) and Western blot (lower panel). Data are representative of three independent experiments. b EZH2 251-374 was phosphorylated by DCAF1, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifying the presence of phosphorylated T367. c EZH2 wild type or T367A mutant proteins were modified by DCAF1 and analyzed by Western blot using the antibody raised against EZH2T367p. Data are representative of three independent experiments. (See also Supplementary Fig. 3). d DCAF1-depleted SW620 cells were infected with lentiviruses expressing 3×FLAG-DCAF1 wild type and K194R kinase-dead mutant as indicated on the top. Cell lysates were prepared and subjected to Western blot analysis with indicated antibodies. Data are representative of three independent experiments. e EZH2-depleted SW620 cells were infected with lentiviruses expressing 3×FLAG-EZH2 wild type, T367A phospho-blocking mutant, or T367D phospho-mimicking mutant as indicated on the top. Aliquots of cell lysates were analyzed by Western blot. Data are representative of three independent experiments. f EZH2-depleted SW620 cells were rescued as in (e), and WST-1 assays were carried out after 3 days of culture. Data are represented as mean ± SEM of three independent experiments. P values were calculated using one-way ANOVA with post-hoc Tukey’s test for multiple comparisons. **P < 0.01 and ***P < 0.001 versus Ctrl sh. (a), (c–f) Source data are provided as a Source Data file.
