Extended Data Fig. 3: USP9X deubiquitylates TK1, thus stabilizing its expression.

(a) Huh7 cells were treated with MG132 (10 μM) for the indicated time. Immunoblotting with the indicated antibodies was performed. (b) Endogenous TK1-depleted Huh7 cells with reconstituted expression of indicated Flag-rTK1 proteins were transfected with HA-Ub and stimulated with or without EGF (100 ng/ml) for 1 h. Cells were harvested with a guanidine-HCl-containing buffer after treated with MG132 (10 μM) for 6 h. Immunoprecipitation and immunoblotting with the indicated antibodies were performed. (c) Selected peptide hits of TK1-associated proteins in Huh7 cells as identified by mass spectrometry were presented. (d) Endogenous TK1-depleted Huh7 cells with reconstituted expression of indicated Flag-rTK1 proteins were stimulated with or without EGF (100 ng/ml) for 1 h. Cells were treated with MG132 (10 μM) for 6 h before harvest. Immunoprecipitation and immunoblotting with the indicated antibodies were performed.

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