Extended Data Fig. 1: CLOCK S106 phosphorylation disassembles the CLOCK–BMAL1 complex.
(b-e, h, j, k, m, n) Immunoprecipitation and immunoblotting with the indicated antibodies was performed. All experiments were repeated at least twice independently. Data are the mean ± SD. (a) The luciferase activity of the indicated cells expressing a Per1-driven luciferase reporter was measured after IGF1 treatment (12 h) (n = 6). *P < 0.01; **P < 0.001 by One-way ANOVA post hoc test. (b, c, m, n) The indicated cells were pretreated with or without the indicated inhibitors for 30 min before IGF1 treatment for 30 min (b, c, m) or transfected with the indicated plasmids before IGF1 treatment for 1 h (n). Total cell lysates and cytosolic fractions were prepared. (d) Flag-CK2α immunoprecipitated from Huh7 cells treated with or without IGF1 for 30 min was incubated with or without CIP for 30 min. (e) Purified GST-CK2α was mixed with or without active His-ERK2 for in vitro kinase assay, followed by incubation with purified His-CLOCK. Proteins precipitated by GST pulldown assay were incubated with or without CIP for 30 min. (f) In vitro kinase assays were performed by mixing purified His–CLOCK with or without purified GST-CK2α in the presence of ATP. Mass-spectrometric analysis was performed. (g) Alignment of CLOCK to the consensus CK2α-phosphorylated substrate motif (SXXD/E). (h, i) Huh7 cells expressing Flag-CLOCK were treated with or without IGF1 for 1 h (h). Immunoblotting (h) or IHC analyses of human HCC samples (i) were performed with the indicated antibodies and a CLOCK pS106-blocking peptide. (j-l) The indicated cells expressing the indicated plasmids (j) were treated with or without IGF1 for 1 h (j-l). Total cell lysates and cytosolic and nuclear fractions were prepared (k). The relative CLOCK abundance was quantified (n = 10) (l). *P < 0.05; ***P < 0.0001 by two-tailed Student’s t test.
