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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Cell Rep. 2014 May 15;7(5):1577–1588. doi: 10.1016/j.celrep.2014.04.027

Figure 5. AKAP79/150 anchoring of both CaN and PKA is necessary for NFAT-dependent transcription.

Figure 5

(A) Modified high K+ stimulation protocol for stimulation of NFAT-dependent transcription. (B) Diagram of the 3xNFAT/AP1-CFPnls transcriptional reporter construct used for single-cell imaging of NFAT-dependent transcription. (C) Summed intensity projection images of neuronal cell bodies and proximal dendrites in NS conditions and 16 hrs after high K+ stimulation (KCl). Neurons were transfected with the 3xNFAT/AP1-CFPnls reporter along with pSilencer empty vector (Control) or 150RNAi plus YFP or the indicated AKAP79-YFP constructs. YFP fluorescence is in white and nuclear-localized CFP fluorescence is in pseudocolor. (D) Quantification of the fold-change in nuclear fluorescence of the 3xNFAT-AP1-CFP-NLS reporter following high K+ stimulation for the indicated conditions. (E) Diagram of the pGL3NFAT plasmid that drives NFAT-dependent transcription of firefly luciferase and the internal transfection control plasmid pRLSV40 driving constitutive transcription of Renilla luciferase. (F) Quantification of NFAT-dependent transcription as normalized luciferase activity measured from lysates of WT and AKAP150 mutant mouse neurons. Data expressed as mean ± SEM. (*p<0.05, **,##p<0.01, and ***p<0.001 by ANOVA with Dunnett post-hoc test; n = 14–49 (rat); n = 6–14 (mouse)). Scale bars = 10μm