Fig. 6: PRMT3 interacts with TEAD4 and P-TEFb to form a transcriptional hub that co-localizes in the nucleus.

a, b HEK293T cells were cotransfected with empty vector or Flag-PRMT3 with Myc-TEAD4 (a), or Myc-TEAD4 with Flag-PRMT3 (b) for 40 h. Flag-PRMT3 (a) or Myc-TEAD4 (b) was immunoprecipitated using anti-Flag antibody or anti-Myc antibody, following with western blot analysis for the indicated protein. c GST pull-down assay was performed using GST-PRMT3 and His-TEAD4 that were purified from E. Coli. The products from the pull-down assay were subjected to western blot analysis for the detection of the indicated proteins. d, e HEK293T cells that were cotransfected with empty vector or plasmid expressing Myc-TEAD4 together with Flag-Tat (d), or plasmid expressing Flag-Tat together with Myc-TEAD4 (e) for 40 hours. Myc-TEAD4 (d) or Flag-Tat (e) was immunoprecipitated using anti-Myc or anti-Flag IP, following with western blot analysis for the indicated protein. f, g Whole cell lysates of HEK293T cells that were transfected with empty vector or plasmid expressing Flag-PRMT3 (f), or plasmid expressing Flag-CycT1 (g) for 40 h were used for anti-Flag IP, following with western blot analysis for the indicated protein. h, i Whole cell lysates of HEK293T cells that were transfected with empty vector or plasmid expressing Myc-TEAD4 (h), or plasmid expressing Flag-CycT1 together with Myc-TEAD4 (i) for 40 h were used for anti-Myc (h) or anti-Flag (i) IP following with western blot analysis for the indicated protein. j–l Whole cell lysates of Hela cells was used for chromatin pellets collection with lysis buffer and immunoprecipitated using anti-IgG, anti-PRMT3, anti-TEAD4, or anti-CDK9 antibody, following with western blot analysis for the indicated protein. m HEK293T cells were cotransfected with plasmids expressing HA-PRMT3 together with EGFP-CycT1 (green), Myc-TEAD4, or EGFP-CycT1 + Myc-TEAD4 for 40 h. Protein localization was detected by immunofluorescence using anti-HA antibodies (yellow) or anti-Myc antibodies (red), and 40,6-diamidino-2-phenyl-indole (DAPI) (nuclei, blue) for confocal microscopy analysis. Scale bars, 10 μm. All western blots are representative of three independent experiments.