At 4 days postinfection, the cells were harvested for

the luciferase assay and an LDH assay after OGD. Primary cortical neurons were infected with the purified adenovirus (adeno-CRE-Luc buy Ipatasertib [firefly] and adeno-TK-Luc [renilla]). The adenovirus containing a CMV promoter driving renilla luciferase was used as an internal control (Promega) as previously described (Katoh et al., 2006). Primary cortical neurons were fixed immediately with 4% PFA for 30 min. For double-immunofluorescence in vivo, free-floating sections (40 μm) were used. See Supplemental Experimental Procedures for details. To prepare an SIK2-Thr484 peptide, a set of oligonucleotides for the SIK2 peptide (Gly479 to Thr489: 5′-gatcGGGCAGCGACGGCACACTCTGTCAGAAGTGACT

Y-27632 nmr and 5′-aattAGTCACTTCTGACAGAGTGTGCCGTCGCTGCCC) was cloned into the BamHI/EcoRI site of the Escherichia coli GST-fusion vector pGEX6P-1, and the GST-SIK2 (Thr484) peptide was purified by using a glutathione-Sepharose column. To prepare active CaMKs, cDNA fragments for constitutive active CaMKs were ligated into the BamH1/NotI site of pEBG mammalian GST-fusion vector, and the active CaMKs were expressed in COS-7 cells followed by purification with a glutathione-Sepharose column. The GST-SIK2 (Thr484) peptide (1 μg) was incubated with CaMKs in a reaction buffer (10 mM Tris-HCl [pH 7.4], 10 mM MgCl2, in the presence or absence of 100 μM ATP) at room temperature for 1 hr, and phospho-SIK2 (Thr484) was detected by western blot analyses with anti-phospho-Thr484 antiserum. These were performed as previously described (Katoh et al., 2006) (see Supplemental Experimental Procedures). Data are representative of at least four independent experiments. Total RNA was extracted by using TRIzol (Invitrogen). cDNAs were prepared by reverse transcription (RT) from total RNA (1 μg) by using SuperScript III and random primers (Invitrogen). One-hundredth of the RT products and standard plasmids were subjected to real-time PCR analyses

with the iQ SYBR Green Supermix (Invitrogen) or TaqMan Probe Universal PCR Master Mix (Applied Biosystems). The specific primers used are listed in Table S1. See oxyclozanide Supplemental Experimental Procedures for details. The generation of Sik2+/− mice has been described by Horike et al. (2010); these mice are now supplied by JCRB Laboratory Animal Resource Bank at the National Institute of Biomedical Innovation (NIBIO) No. nbio071; http://animal.nibio.go.jp/index.html. Although Sik2+/− ES cells were established in C57BL/6-derived cells (RENKA), Sik2+/− mice were mated with C57BL/6J mice for ten generations; then, mouse colonies were amplified for the experiments. The experimental protocols using mice were approved by committees at the NIBO.