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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Nat Med. 2010 Jul 18;16(8):872–879. doi: 10.1038/nm.2181

Figure 5.

Figure 5

CIB1 facilitates calcineurin/NFAT activation. (a) Neonatal rat cardiac myocytes were infected with AdNFAT-luc and Adβgal, AdCIB1 or AdCain and stimulated with PE (10 μM) for 48 hrs as indicated. NFAT-dependent luciferase activity was measured and normalized to total protein content. N=6–15 per condition. *p<0.001 vs. Adβgal and Adβgal + PE, #p<0.001 vs. AdCIB1 + PE. (b) Neonatal rat cardiac myocytes were infected with AdNFAT-luc and Adβgal, AdCIB1 or AdCIB1Δ and stimulated with PE for 48 hrs as indicated. N=5–15 per condition, *p<0.001 vs. Adβgal and Adβgal + PE, #p<0.001 vs. AdCIB1 + PE. (c) Calcineurin phosphatase assay over 30 minutes against the phosphorylated RII peptide with recombinant calcineurin holoenzyme (2.5 μg = 20 units) and CIB1 (5 μg). (d) Neonatal rat cardiac myocytes were infected with AdNFAT-luc and transfected with control or CIB1 siRNA, then stimulated with PE for 24 hrs as indicated. N=6 per condition, *p<0.001 vs. control siRNA, #p<0.001 vs. control siRNA + PE. (e) NFAT luciferase activity (normalized to total protein content) measured from NFAT-luc reporter transgenic mice WT or null (−/−) for Cib1 after sham and TAC surgery as indicated. N=3–6 mice per condition, *p<0.05 vs. WT sham. (f) Immunoblot for the indicated proteins from WT or Ppp3r1fl/fl mouse embryonic fibroblasts (MEFs) infected with Adβgal control or AdCre to delete CnB1 protein if appropriate. (g) Immunoblot for the α1C subunit of the L-type Ca2+ channel and CIB1 after IP from CIB1 overexpressing mouse hearts 2 weeks after TAC or sham operation. CIB1 antibody or control IgG was used for the IP. (h) Immunoblot against the α1C subunit of the L-type Ca2+ channel after a pull-down assay from neonatal cardiomyocyte lysate incubated with the indicated GST-CIB1 fusion constructs. (i) Neonatal rat cardiac myocytes were transfected with control or CIB1 siRNA and infected with AdNFAT-luc, then stimulated with PE and verapamil (10μM) for 24 hours as indicated. N=4–6 per condition, *p<0.05 vs. control siRNA, #p<0.001 vs. control siRNA + PE.