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. Author manuscript; available in PMC: 2017 May 21.
Published in final edited form as: Oncogene. 2016 Nov 21;36(16):2319–2327. doi: 10.1038/onc.2016.397

Figure 3. EP4 activates cAMP signaling and ligand-independent ER activation through CARM1.

Figure 3

(a) Western blot analysis of AKT, mTOR (S6 kinase), and MAPK (ERK1/2) pathways in MCF7-LTED cells treated with vehicle or EP4 antagonists. (b) Dose response curve measuring induction of cAMP by EP4 agonist (L-902,688) in MCF7-LTED cells treated with 10nM EP4-antagonist (ONO-AE1-329) or vehicle. (c) Immunoblot of CARM1 in MCF7 and MCF7-LTED cells treated with EP4 antagonist or vehicle in the absence of estrogen. CARM1 immunoprecipitation (IP) followed by western with antibodies for phosphorylated PKA substrate, ERα or CARM1. (d–e) ChIP-qPCR of ERα targets (d) pS2 and (e) PGR with either no antibody (noAB), CARM1 antibody, or ERα antibody in MCF7-LTED cells treated with EP4 agonist or vehicle. Chr8q24 is a gene desert negative control region. (f) Proliferation of MCF7 LTED cells treated with CARM1 siRNA relative to MCF7 LTED cells treated with negative control siRNA. (g) RT-qPCR analysis of CARM1 expression in MCF7 LTED CARM1 knockdowns from (f). * is p-value < 0.05, ** is p-value < 0.01, and *** is p-value < 0.001. Error bars are s.e.m. of three replicates. IB: immunoblot. p-: phosphorylated.