Fig. 3: FBXW7 negatively regulates CHD6 stability.

a Sequence alignment of the putative FBXW7-recognized degron on CHD6. [LIVMP] refers to a group of amino acids, Leu, Ile, Val, Met, Pro, are all allowed; {0,2}, 0, 1, and 2 positions of any kind of amino acids; (T), phosphorylated Thr;.., any kinds of amino acids. b Representative immunoblots showing CHD6 steady-state expression in HCT116 cells upon FBXW7 overexpression or KD. c The interaction between exogenous FBXW7 and CHD6 was determined by co-IP assay. 293T cells were transfected with Myc-tagged CHD6 and Flag-tagged FBXW7. The cell lysates were pulled down with Myc-tagged magnet beads and immunoblotted with the indicated antibodies. WCL: whole cell lysates. d The interaction between endogenous FBXW7 and CHD6 was determined by co-IP assay. HCT116 cell lysates were pulled down with an anti-CHD6 antibody and immunoblotted with FBXW7. e Representative immunoblots showing CHD6 protein turnover rate in HCT116 cells, with or without FBXW7 overexpression (left). Quantification of CHD6 turnover rate (right). Cells were transfected with the indicated plasmids in the presence of CHX (60 μg/mL) for the indicated times. CHD6 levels were normalized to Vinculin and then normalized to the t = 0 control. f 293T cells were transfected with the indicated plasmids and treated with MG132 (10 μM) 6 h before harvest. The cell lysates were pulled down with nickel beads and immunoblotted with an anti-Myc antibody. g, h Representative immunoblots showing the turnover rate of Flag-tagged CHD6 WT or Flag-tagged CHD6 T2127A/T2131A, with or without Myc-tagged FBXW7 overexpression in 293T cells (g). Quantification of CHD6 turnover rate (h). Transfected cells were treated with CHX (60 μg/mL) for the indicated times. Flag-tagged CHD6 levels were first normalized to Vinculin, and then to the t = 0 control. i 293T cells were transfected with the indicated plasmids and treated with MG132 (10 μM) 6 h before harvest. The cell lysates were pulled down with nickel beads and immunoblotted with an anti-Flag antibody. j 293T cells were transfected with the indicated plasmids. The cell lysates were immunoblotted with the indicated antibodies. k Representative immunoblots showing the turnover rate of Flag-tagged CHD6 WT or Flag-tagged CHD6 P2128L, with or without Myc-tagged FBXW7 overexpression in 293T cells (left). Transfected 293T cells were treated with CHX (60 μg/mL) for the indicated times. Quantification of CHD6 WT and P2128L mutant turnover rate (right). Flag-tagged CHD6 levels were first normalized to Vinculin, and then to the t = 0 control.