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. 2016 May 26;44(17):8129–8143. doi: 10.1093/nar/gkw483

Figure 5.

Figure 5.

G9a associates with E2F1 and increases PCAF occupancy at E2F1 target promoters. (A) EGFP-G9a, Flag-PCAF and E2F1 were transfected in 293T cells. PCAF was immunoprecipitated using anti-Flag beads. Interaction with G9a, E2F1 and PCAF was analysed by western blot. Expression of G9a, PCAF and E2F1 was analysed in lysates (input). (B) Endogenous G9a was immunoprecipitated from C2C12 cells using nuclear extracts (left panel). Association with E2F1 and PCAF was tested by immunoblotting with anti-E2F1 and anti-PCAF antibodies. M denotes lane with molecular weight markers. In reverse IP experiments, endogenous E2F1 was immunoprecipitated and the association with G9a and PCAF was tested (right panel). (C) Immunoprecipitation was performed with anti-G9a antibody (first IP) and checked for association with E2F1 and PCAF. Elute from the first IP was re-immunoprecipitated (second IP) with anti-E2F1 antibody and immunoblotted with anti-G9a and anti-PCAF antibodies. The results are representative of at least two independent experiments. (D) pBabe and pBabe-G9a cells (left panel) as well as siRNA and siG9a C2C12 cells (right panel) were transfected in triplicates with 200 ng of a wild-type cyclin D1 reporter (pD1luc) or pD1lucΔE2F1 that harbours point mutations at E2F1 binding site. Luciferase activity was measured using dual luciferase reporter assays. (E) G9a overexpressed (left panel) and knockdown cells (right panel) were transfected in triplicates with 200 ng pD1luc in the absence and presence of PCAF (50 ng) and luciferase activity was measured. Results are representative of at least two independent experiments. Error bars indicate the mean luciferase activity from triplicate transfections in each experiment ± SD. (F) Endogenous E2F1 was immunoprecipitated from pBabe and pBabe-G9a cells grown in growth medium (Day 0). Interaction was examined by immunoblotting with anti-PCAF, anti-Rb1 and anti-E2F1 antibodies. (G) ChIP assays were performed with anti-E2F1 and anti-PCAF antibodies at CyclinD1 and DHFR promoters in proliferating pBabe and pBabe-G9a cells. Results are representative of two independent experiments. Error bars indicate the mean of triplicate q-RT-PCR in each assay ± SD.