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. 2017 Aug 8;114(34):E7121–E7130. doi: 10.1073/pnas.1704999114

Fig. 5.

Fig. 5.

Phosphorylation of EcRB1 promotes an interaction between EcRB1 and USP1 as well as gene expression in HaEpi cells. (A) Identification of the phosphorylation site in EcRB1 by determining the number of moles of phosphorus per mole of EcRB1-His or its mutants (T176A, T468A, and S510A) overexpressed in cells. EcRB1-His and site mutants of the protein were overexpressed in cells for 48 h, followed by treatment with 20E or an equivalent amount of DMSO. (B) Western blot analysis confirmed that the site of phosphorylation in EcRB1 was Thr468, under the conditions described in A. β-Actin was used as a loading control. Statistical analyses of the density of Western blotting bands were performed with Quantity One software. (C) ChIP analyses of the EcRE-binding abilities of EcRB1-RFP-His and EcRB1-T468A-RFP-His in the HR3 promoter region via anti-RFP precipitation and qRT-PCR analysis. 20E (2 µM) or DMSO treated the transfected cells for 3 h. Results are presented as percentage of enrichment relative to input. Input, sample before immunoprecipitation. Enrichment relative to input = (sample precipitated by antibody – sample without antibody)/input × %. lgG, nonspecific mouse lgG. Primer EcRE, primers targeted to EcRE. Primer HR3, primers targeted to HR3 ORF. (D) Phosphorylation of EcRB1 T468 promoted the interaction of EcRB1 with USP1 under 20E induction. The proteins were extracted from transfected cells after treatment with 2 µM 20E or DMSO for 3 h. The input showed the levels of the USP1-GFP-His, EcRB1-RFP-His, and EcRB1-T468A-RFP-His proteins expressed in the cells. β-Actin was used as a loading control, and the proteins were detected via Western blot analysis following SDS/PAGE using gels with a concentration of 7.5%. The phosphorylation of USP1 and EcRB1 was not detected due to the GFP and RFP tags increasing the molecular mass. Nonspecific mouse lgG as a negative control. (E) Comparison of HR3 expression levels in RFP-His–, EcRB1-RFP-His–, and EcRB1-T468A-RFP-His–overexpressing cells under 20E induction. pIEx-4-RFP-His, pIEx-4-EcRB1-RFP-His, and pIEx-4-EcRB1-T468A-RFP-His plasmid-transfected cells were treated with 20E (2 µM) for 6 h. An equivalent amount of diluted DMSO was added as a control. Statistical analyses were performed using Student’s t test (*P < 0.05, ***P < 0.001), and the values are the mean ± SD. Three biological replicates and three technical replicates were performed for A–E.