Fig. 4. CK1α promotes the turnover of CBX4 by CHIP.

a, c HEK293T cells were cotransfected with the indicated plasmids for 48 h and then subjected to IP using anti-FLAG antibody followed by Western blotting analysis of three independent experiments. b HEK293T cells cotransfected with the indicated plasmids for 36 h were incubated with 20 μg/ml CHX for the indicated periods and then analyzed by Western blotting. Quantitation of Flag-CBX4 protein levels was based on the Western blotting results. n = 1. d U2OS/MTX300 cells were subjected to IP using anti-CBX4 antibody or anti-IgG antibody followed by Western blotting as indicated. e HEK293T cells cotransfected with the indicated plasmids for 48 h were analyzed by Western blotting. f HEK293T cells cotransfected with the indicated plasmids for 40 h were incubated with 10 μM MG132 for 8 h and then subjected to IP using anti-FLAG antibody followed by Western blotting. g Flag-CBX4 WT or T437A mutant was purified from HEK293T cells, incubated with or without the purified V5-CK1α in vitro as described in Methods, and then analyzed by Western blotting. h, i The co-IP assay was performed using the indicated U2OS/MTX300 stable cells with anti-CBX4 antibody or anti-IgG antibody as indicated. j–m The indicated cells were starved by excluding fetal bovine serum (FBS) from the medium for 24 h, and then the cells were treated with TNFα, RANKL, and M-CSF for 24 h as indicated and subjected to Western blotting (j–l) or the co-IP assay using anti-CBX4 antibody or anti-IgG antibody as indicated (m). These resluts are repeated of three independent experiments. Source data are provided as a Source Data file.