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. 2012 Feb 10;31(7):1785–1797. doi: 10.1038/emboj.2012.17

Figure 4.

Figure 4

Functional properties of arginine methylated E2F-1. (A) Transcription properties of WT E2F-1 and the KKK mutant. U2OS cells were transfected with expression vectors encoding WT E2F-1 or the KKK mutant, together with p73-luciferase, Cdc6-luciferase, E2F-1-luciferase, Apaf1-luciferase, DHFR-luciferase or cyclin E-luciferase for 48 h as indicated, and pCMV-βgal to monitor transfection efficiency. Relative luciferase activity (luciferase/βgal) is shown together with the expression level of the ectopic proteins underneath; n=3. (B) ChIP of U2OS cells transfected with expression vectors encoding HA-tagged WT E2F-1 or the KKK mutant as described, followed by immunoprecipitation with anti-HA on the E2F target genes, and quantitation by real-time PCR shown in (ii); n=4. (C) ChIP of U2OS cells transfected with PRMT5 (P) or control (C) siRNA followed by immunoprecipitation with anti-E2F-1 antibodies on the indicated E2F target genes, and quantitation by real-time PCR (i); the level of the input protein is shown below (ii); n=2. (D) Effect of PRMT5 siRNA on E2F-1 RNA: PRMT5 (P) or control (C) siRNA was transfected into U2OS cells in the presence or absence of etoposide (Et) and cells harvested at 72 h post-transfection. RNA levels for E2F-1, 18S and GAPDH were assayed as indicated. (E) Effect of PRMT5 (P) or control (C) siRNA on E2F-1 and p73 RNA at 72 h post-transfection and quantitation by real-time PCR.