Bmal1 antibody

Two canonical E-box sequences (CACGTG) were identified at 2016 and 646 bp upstream of the 5′ transcription start site of the Stxbp1 gene. ChIP was conducted using the SimpleChIP Enzymatic Chromatin IP Kit with Magnetic Beads #9003 (Cell Signalling) according to the manufacturer’s instructions and as previously described (10 (link)). Briefly, mGLUTag L-cells were fixed at 8 and 20 hours after cell synchronization, followed by DNA digestion and incubation with Bmal1 antibody (Cell Signaling Technology). Protein was precipitated using protein G magnetic beads and DNA was eluted and quantified by RT-quantitative PCR (RT-qPCR) (Table S2 in File Inventory (29) ). Signal from each immunoprecipitation was calculated as a percent of the total input chromatin using the following formula: Signal relative to input=2%×.

A construct to knock out
BMAL1 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA; BMAL1 CRISPR/Cas9 KO Plasmid, Santa Cruz sc-419206). The BMAL1 CRISPR/Cas9 KO plasmid is a pool of three different gRNA plasmids with the following sequences: sc-419206 A: Sense: 5′- TAGATAAACTCACCGTGCTA-3′; sc-419206 B: Sense: 5′-CTGCACGTACCCTGAGAATT-3′; sc-419206 C: Sense: 5′-TTACTAGGTACCTTCCATGA-3′. The
Per2-luc 661W cells cultured on a 12-well plate were co-transfected with 0.5 μg of the BMAL1 CRISPR/Cas9 KO plasmid and 0.5 μg of the BMAL1-R HDR plasmid (Santa Cruz sc-419206-HDR). The plasmid included a puromycin resistance gene (used for the selection of colonies) and the red fluorescent protein (RFP) to detect the correct insertion of the plasmid in the genome. After selection in a medium containing puromycin (4 μg/mL), only cells with RFP signals were isolated. Then the removal of
BMAL1 from the 661W cells was verified by western blotting using the BMAL1 antibody (Cell Signaling Technology, Danvers, MA, USA; cat. no. 14020; RRID:AB_2728705).