Figure 1
From: Loss of the conserved PKA sites of SIK1 and SIK2 increases sleep need
SIK family mRNA expression and mutant proteins. (a) Scheme of SIK1, SIK2, and SIK3. The serine residue in the PKA consensus sequence is conserved among the family. Although there are multiple protein isoforms of SIK3, this scheme shows the longest isoform. (b) Digital PCR results of Sik1, Sik2 and Sik3 mRNA of the cerebral cortex, hippocampus, hypothalamus, liver and brown adipose tissue (BAT) of the wild-type mice (n = 4). Each sample was measured in duplicate. One-way analysis of variance followed by Tukey’s test. (c-e) In situ hybridization of Sik1 and Sik2. (c) Sik1 mRNA was strongly expressed in the suprachiasmatic nucleus (SCN) and broadly expressed in the forebrain. Scale bar, 500 μm. (d) In situ hybridization showed that Sik2 mRNA was broadly expressed in the forebrain. Scale bar, 500 μm. (e) Sik1 and Sik2 were expressed in the hippocampal dentate gyrus of the wild-type mice (upper and middle). Sik2 expression was not detected in the dentate gyrus of the Sik2-deficient mice (lower). Scale bars, 100 μm. (f-h) Sik1 (f), Sik2 (g) and Sik3 (h) mRNA expression in the cerebral cortex, hypothalamus, BAT, liver and adrenal gland after one week of high-salt diet feeding. Two-tailed t-test with Bonferroni correction. (i) SIK2 protein was expressed in the BAT of the Sik2+/+ and Sik2S587A/S587A mice. SIK2 was not detected in the BAT of the Sik2-deficient mice. (j) FLAG-SIK1 WT, FLAG-SIK1 S577A, FLAG-SIK2 WT, FLAG-SIK2 S587A, FLAG-SIK3 WT, and FLAG-SIK3 S551A transiently expressed in HEK293 cells were immunoprecipitated with an anti-FLAG antibody and then subjected to immunoblotting using anti-FLAG, anti-phospho-PKA substrate motif, and anti-14-3-3 antibodies. BNST, bed nucleus of the stria terminalis; MPO, medial preoptic area; och, optic chiasma; PVT, paraventricular thalamic nucleus. One-way ANOVA followed by Tukey’s test. Data are presented as the mean ± SEM. group. Full blots are shown in the Supplementary Information.
