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. 2009 Aug 26;4(8):e6766. doi: 10.1371/journal.pone.0006766

Figure 6. Over-expression of CARM1 restores iAs-mediated transcriptional repression.

Figure 6

(A) Cells were transfected with pSG5-HA-CARM1, pSG5-GRIP1/FL or a combination of the two expression plasmids at indicated concentrations and treated with 5 nM Dex±8 µM iAs for 24 h. CAT activity was measured and expressed as percent conversion from unacetylated to acetylated chloramphenicol. Representative of an experiment repeated 3 times n = 3 replicate points. (B) Western blot analysis of proteins from cells transfected with 0.5 µg of pSG5-HA-CARM1 or pSG5-GRIP1/FL plasmid or non-transfected cells (NT), incubated with antibodies to CARM1, GRIP1 and b-actin. The HA-tagged CARM1 migrates slightly higher in the gel than endogenous CARM1. (C) Cells transfected with pSG5-HA-CARM1 at the indicated concentrations or pSG5-HA-EMPTY at 1.5 µg and treated for 2 hours with 5 nM Dex±8 µM iAs. SGK mRNA was measured by RT-qPCR corrected for actin mRNA. The values indicated on the y-axis are RT-qPCR Units. Experimental points are in triplicate and the experiment was repeated twice. (D) pSG5-GRIP1/FL or Empty vector was over-expressed in cells at the concentrations indicated and SGK mRNA was measured by RT-qPCR and analyzed as in (C).