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. 2021 Apr 7;7(15):eabe2261. doi: 10.1126/sciadv.abe2261

Fig. 1. EZH2 increases FOXA1 protein stability through physical interaction.

Fig. 1

(A and B) PCa cells were infected with control (shCtrl) or two independent shEZH2 lentiviruses (shEZH2-C and shEZH2-3′) for 72 hours and subjected to WB. (C) LNCaP cells were infected with the indicated EZH2 lentivirus for 96 hours and analyzed by WB. (D) LNCaP cells were treated with protein synthesis inhibitor CHX (150 μg/ml) for 0, 2, 4, and 6 hours before they were collected for WB. (E and F) 293T cells were transfected with indicated plasmids for 48 hours. Coimmunoprecipitation (co-IP) was performed using a hemagglutinin (HA) (E) or FLAG antibody (F), followed by WB. (G) Co-IP with FOXA1, EZH2, or immunoglobulin G (IgG) antibody in LNCaP nuclear lysates followed by WB. (H) Purified recombinant GST–green fluorescent protein (GFP) or GST-EZH2 coupled to GST beads were used to pull-down purified MBP-FOXA1, which was analyzed by WB using an MBP antibody. (I) Nuclear extracts from LNCaP cells were fractionated and subjected to WB. (J) Co-IP using LNCaP nuclear lysates was performed with FOXA1, SUZ12, or IgG antibody. (K and L) EZH2 was cotransfected into 293T cells along with various FLAG-tagged FOXA1 domain constructs (K). Co-IP assay was performed with anti-FLAG M2 beads followed by WB using anti-EZH2 (L). EZH2-binding domains are highlighted in yellow.