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. 2004 Dec 16;24(1):85–96. doi: 10.1038/sj.emboj.7600500

Figure 7.

Figure 7

Enzymatic activities of CARM1 and p300/CBP are required for full cooperativity between CARM1 and p300. (A) Carm1(+/+) MEF cells (black bars) and Carm1(−/−) MEF cells (dashed bars) were cotransfected with pGL3-MIP-2(−770), pGL3-IP-10(−533) or pOLUC-IL-6(−303/+23) (5 μg) and pphRSVnt-β-gal (200 ng) alone or together with CMV-CARM1 (200 ng) or CMV empty vectors as indicated. Cells were subsequently stimulated with TNFα (10 ng/ml) for 6 h. Cells were harvested 32 h after transfection and the indicated activation of NF-κB-dependent gene expression determined as described in Figure 1. (B, C) Enzymatic activities of CARM1 and p300/CBP are required for full cooperativity between CARM1 and p300. Carm1(−/−) MEF cells were cotransfected with pGL3-MIP-2(−770) (B) or pGL3-IP-10(−533) (C) (5 μg) and pphRSVnt-β-gal (200 ng), together with CMV-p65 (80 ng) CMV-CARM1-WT or CARM1-E267Q (200 ng), CMV-p300-WT or p300mutAT2 (750 ng) and CMV empty vectors as indicated. Cells were harvested 32 h after transfection and the indicated activation of NF-κB-dependent gene expression determined as described in Figure 1. (D) Both wild type and mutant forms of CARM1 and p300 interact to the same extent with p65 in vivo. 293T cells were transfected with CMV expression vectors, either for wild type or enzymatic mutant forms of myc tagged p300 (left panel) or CARM1 (right panel) as well as pphCMV-kozak-myc empty vector. p65, CARM1 and p300 were coimmunoprecipitated (IP) from nuclear extract of TNFα-treated 293T cells (30 min, 10 ng/ml) using an anti-myc antibody. Bound proteins were resolved by SDS–PAGE and subsequently detected by immunoblot (IB) analysis for p65, CARM1 and p300. Input lanes represent 10% of the input.