Extended Data Figure 4: PTEN–AKT–GSK3β pathway promotes CHD1 degradation through β-TrCP-mediated ubiquitination–proteasome pathway.

a, HA-tagged ubiquitin (HA–Ub) was transfected into 293T cells for 40 h, followed by 6 h of MG132 treatment and immunoprecipitation (IP) of endogenous CHD1. CHD1 and HA were detected by immunoblot. b, Conservation of two β-TrCP binding motifs in vertebrates. c, d, Immunoblots of CHD1 in BPH1 cells overexpressing Flag-tagged β-TrCP or knockdown of β-TrCP (Yap as positive control). e, HA–Ub and siBTRC (targeting β-TrCP) were transfected into 293T cells for 48 h, followed by 8-h of treatment with MG132 treatment (10 μM) and detection of ubiquitin-bound CHD1 by immunoblot. f, V5-tagged wild-type (WT) or two β-TrCP binding motif mutants (DSGXXS ≥ DAGXXA) of CHD1 were introduced into BPH1 cells, followed by CHX treatment over a time course, and V5-tagged CHD1 was detected by immunoblot. g, h, V5-tagged wild type or the two β-TrCP-binding-motif mutants of CHD1 were introduced into BPH1 cells, followed by V5-immunoprecipitation and detection of ubiquitination and β-TrCP binding by immunoblot. i, Schematic diagram of GSK3β substrate consensus sequences in β-TrCP binding motifs of CHD1. j, Endogenous CHD1 was immunoprecipitated, followed by immunoblot using GSK3β antibody. k, LNCaP cells overexpressing PTEN were treated with 2 μM CHIR for 24 h, and CHD1 protein levels were detected by immunoblot. β-actin was used as a loading control. Representative data of triplicate experiments are shown.