Extended Data Fig. 2: ERK-mediated TK1 S13/231 phosphorylation stabilizes TK1.
Immunoprecipitation and immunoblotting with the indicated antibodies were performed. (a) Huh7 cells stably transfected with or without Flag-TK1 were serum-starved and treated with or without EGF (100āng/ml) for the indicated periods of time. (b) Protein sequences of TK1 and the ERK1/2 recognized motif (red labeled) predicted by Scansite. (c) Immunohistochemical analyses of human HCC samples were performed with the indicated antibody in the presence or absence of a blocking peptide for TK1 pS231. Representative images from three independent experiments are shown. (d) Endogenous TK1-depleted Huh7 cells were reconstituted with WT Flag-rTK1, Flag-rTK1 S13A/S231A or Flag-rTK1 V23A/L25R/L137R/L139R and then stimulated with or without EGF (100āng/ml). (e, f) HCC cells were stably transfected with WT Flag-TK1 and then pre-treated with or without U0126 (10āĪ¼M), RO-3306 (5āĪ¼M) for 30āmin before EGF (100āng/ml) treatment for 2āh. (g) Endogenous TK1-depleted Huh7 cells were reconstituted with WT Flag-rTK1, Flag-rTK1 S13D/S231D and then stimulated with or without CHX (100āng/ml) for the indicated periods of time. Quantification of TK1 levels initial protein levels is shown. Data represent the meanāĀ±āSD of three biologically independent experiments analyzed by two-way ANOVA.
