Fig. 10

RCAN1 expression is normally arrhythmic in young mice. A Representative western blot images of RCAN1 and BMAL1 in the hippocampi of Rcan1 WT adult mice (3–6 months old) at ZT11, ZT17, ZT23, and ZT5. RCAN1 isoforms: RCAN1.1L (~38 kDa), RCAN1.1S (~28 kDa), and RCAN1.4 (~28 kDa). β-tubulin, loading control. B Densitometric measurements of RCAN1 isoform abundance normalized to β-tubulin levels are displayed as percentages of the mean relative optical density (OD) in ZT11 hippocampi. There were no temporal variations in RCAN1.1L and RCAN1.1S/1.4 levels at the time points assessed. N = 6 ZT11, 7 ZT17, 6 ZT23, 6 ZT5 mice. C Densitometric measurements of BMAL1 abundance normalized to β-tubulin levels are displayed as percentages of the mean relative OD in ZT11 hippocampi. BMAL1 levels were significantly elevated at ZT23 compared with ZT11. N = 6 mice per time point. D Representative image of the SCN (dashed white outline) in WT coronal mouse brain sections co-stained for RCAN1 (red), the nuclear marker Hoechst (blue), the clock protein BMAL1 (green), and the neuronal marker NeuN (white). RCAN1 was present throughout the SCN. RCAN1 signal specificity was confirmed with Rcan1 KO SCN sections. Scale bar = 200 μm. E Higher magnification shows that RCAN1 is expressed diffusely in the SCN, with some signal colocalizing with a subset of BMAL1-positive cells (arrows). Scale bar = 20 μm. F Representative western blot images of RCAN1 and BMAL1 at ZT11 and ZT23 in the SCN of Rcan1 WT and KO mice (3–6 months old) and G WT, Dp16, Dp16/Rcan1^2N (D/R^2N) mice (3–6 months old). H Densitometric measurements of RCAN1 isoform abundance normalized to β-tubulin levels in the SCN of Rcan1 KO mice and I Dp16 and Dp16/Rcan1^2N mice are displayed as percentages of the mean relative OD in respective WT SCN controls at ZT11. RCAN1 levels were affected by genotype but did not differ at the time points assessed. J Densitometric measurements of BMAL1 abundance normalized to β-tubulin levels in the SCN of Rcan1 KO mice and K Dp16 and Dp16/Rcan1^2N mice are displayed as percentages of the mean relative OD in respective WT SCN controls at ZT11. BMAL1 levels in the SCN were not significantly different at the time points assessed in WT mice or compared to Dp16 and Dp16/Rcan1^2N mice but were significantly elevated at ZT23 in Rcan1 KO mice compared with WT littermates. F–K N = ZT11: 7 Rcan1 WT, 5 Rcan1 KO, 7 WT (Dp16 strain), 6 Dp16, 7 Dp16/Rcan1^2N mice; ZT23: 5 Rcan1 WT, 6 Rcan1 KO, 8 WT (Dp16 strain), 6 Dp16, 6 Dp16/Rcan1^2N mice. All data are mean ± S.E.M. *p < 0.05; ****p < 0.0001