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. Author manuscript; available in PMC: 2013 Mar 5.
Published in final edited form as: Nat Cell Biol. 2012 Sep 16;14(10):1105–1112. doi: 10.1038/ncb2578

Figure 4.

Figure 4

WFS1 forms an active complex with AC8. (a) cAMP measured in Wfs1+/+ (WT) and Wfs1−/− (KO) islets treated with 2.5 mM glucose (2.5G), 16.7 mM glucose (16.7G) or 16.7G with: 100 nM GLP-1, 0.5 mM IBMX, GLP-1 + IBMX, 100 µM glyburide, 10 µM forskolin (FSK) and 30 mM KCl. cAMP was normalized to total protein (n = 3). **P < 0.01, ‡P < 0.001 when compared with WT. (b) Insulin release measured in a including 1 mM DBcAMP and normalized to insulin content (n = 3). *P < 0.05, **P < 0.01, ‡P < 0.001 when compared with WT. (c) Total adenylyl cyclase (AC) activity measured in rat islets transduced with GFP or WFS1 lentivirus treated for 2 h with 2.5G or 16.7G (n = 3). *P < 0.05, **P < 0.01 when compared with GFP (16.7G). (d) AC8 immunoprecipitated (IP) from INS-1 832/13 pTetR cells overexpressing WFS1, treated with 2.5G or 16.7G for 1 h. Immunoprecipitates immunoblotted (IB) with anti-WFS1, anti-AC8, anti-calmodulin (CaM) and anti-actin. (e) Quantification of 16.7G IP proteins as fold increase from 2.5G (dotted line) expressed in arbitrary units (a.u.; n = 3). *P < 0.05, **P < 0.01 when compared with WT. (f) AC1 IP from INS-1 832/13 treated the same as in d. Immunoprecipitates IB with anti-WFS1, anti-AC1 and anti-actin antibodies (n = 3). (g) Quantification of proteins the same as in e. **P <.01 when compared with 2.5G. (h) CaM IP from INS-1 832/13 pTetR cells expressing shRNA against Wfs1 (shWFS1) ± expression of WFS1 mutant P724L (MUT) and treated as in d. Immunoprecipitates were IB the same as in d. (i) Quantification of 16.7G IP proteins the same as in g (n = 3). *P < 0.05, **P < 0.01 when compared with 2.5G. (j) AC8 IP from the same cells as in a and treated with 16.7G ± 1 µM Tg for 2 h. Immunoprecipitates IB the same as in d. (k) Quantification of IP proteins expressed in a.u. (n = 3). *P < 0.05 when compared with control (Tg). (l) Insulin release in the same cells as in d transfected with scramble shRNA (Scr) or shRNA against Ac8 (shAC8), and treated with doxycycline to ectopically express WFS1 (Scr/WFS1 and shAC8/WFS1). Insulin normalized to insulin content (n = 3). *P < 0.05, **P < 0.01 when compared with Scr (16.7G). All data are means ± s.d. Uncropped images of blots are shown in Supplementary Fig. S6.