Ab3517

CHiP was performed using SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) (Cell signaling, Tokyo, Japan, #9002). Isolated IECs or cultured cells (HCT116 and Caco-2) were cross-linked in 1% formaldehyde, followed by the CHiP assay according to the manufacturer’s protocol. Rabbit polyclonal antibodies to CLOCK (ab3517) and BMAL1 (ab3350) were from Abcam (Cambridge, UK). DNA was purified and Q-PCR was done using SYBRgreen reagent (QIAGEN, Hilden, Germany). Primers used were as follows: mouse Occludin promoter forward 5′-CTCCCATCCGAGTTTCAGGT-3′ and reverse 5′-GCTGTCGCCTAAGGAAAGAG-3′, mouse Claudin-1 promoter forward 5′-GTTTGCAGAGACCCCATCAC-3′ and reverse 5′-AGAAGCCAGGATGAAACCCA-3′, human Occludin promoter forward 5′-CTCCCATCCGAGTTTCAGGT-3′ and reverse, human Claudin-1 promoter forward 5′-CTCCCCGCCTTAACTTCCTC-3′ and reverse 5′-CAGGAAGGCGAGAATGAAGC-3′, 5′-GGAGTGTAGGTGTGGTGTGT-3′.

IECs were lysed in buffer containing 0.1 M Tris-HCl (pH 7.5)/2% SDS/10% glycerol/5% 2-mercaptoethanol, boiled at 95°C for 5 min and centrifuged at 15000 rpm for 5 min and analyzed by reducing SDS-PAGE. Immunoblots were performed using antibodies against Claudin-1 (Abcam, Cambridge, UK, ab15098, 1∶100), Occludin (Applied Biosystems, 71–1500, 1∶250), Clock (Abcam, ab3517, 1∶200), Bmal1 (Abcam, ab3350, 1∶200) and β-actin (Santa Cruz, Texas, sc-1615, 1∶200). Quantitative analysis of Western blotting was done by using the Scion Image Software package and relative intensities of the target proteins to β-actin were shown.

The following antibodies were used: polyclonal rabbit anti-CLOCK (ab3517, Abcam, (Cambridge, UK)), polyclonal rabbit anti-BMAL1 antibody (NB100-2288, Novus Biologicals), monoclonal mouse anti-GFAP (#3670, Cell signaling technology (Danvers, MA, USA)), polyclonal rabbit anti-Caspase-3 (#9662, Cell signaling Technology (Danvers, MA, USA)), and monoclonal mouse anti-β-actin (A5316, Sigma-Aldrich (St Louis, MO, USA)). Fluoroshield™ with DAPI (F6057, Sigma-Aldrich (St Louis, MO, USA)) was used for nuclear staining and mounting. Sections were mounted onto gelatin-coated slides with Canada Balsam (Wako, Tokyo, Japan) following dehydration.

ChIP assay was performed as previously described21 (link). In brief, nuclear fractions were extracted as chromatin from sonicated cells. After preclearance with protein G agarose/salmon sperm DNA (Millipore, Billerica, MA), chromatin fractions were incubated with anti-mouse/human KAT13D/CLOCK antibody ChIP grade (rabbit polyclonal IgG) (ab3517, Abcam, Cambridge, MA), anti-mouse SLC22A3 (OCT3) Ab (ab191446, Abcam, Cambridge, MA) or control purified rabbit IgG (no. 026102, Invitrogen, Inc., Carlsbad, CA). The “input” samples were DNA extracts prepared from untreated chromatin, and DNA was extracted from immunoprecipitated chromatin. Equivalent masses of immunoprecipitated and input DNA were analyzed by real-time PCR using primers and a TaqMan probe for the promoter region of OCT3 (Supplementary Fig. S7), mPer2-E2, and mPer2-E5 (ref. 22 (link)) as follows:
OCT3Sense (5′-GGAGCTGGAGGAATGTGATGAC-3′),
Antisense (5′-CTCCAGGAACAGAGTTTCATACCT-3′),
TaqMan probe (5′-FAM-CTGCACCTGCACCTGC-MGB-3′).
mPer2-E2,
Sense (5′-CCACCAATTGATGAGCGTAGC-3′),
Antisense (5′-CGTCGCCCTCCGCTG-3′),
TaqMan probe (5′-FAM-TCACGTTTTCCACTATGTG-MGB-3′).
mPer2-E5,
Sense (5′-TCCTGCCACATTGAGATTTGG-3′),
Antisense (5′-GTGATTGCCCCACACTCACA-3′),
TaqMan probe (5′-AAGAGATGGCACGTTAGT-MGB-3′).
Data are presented as the ratio of the cycling threshold value of immunoprecipitated DNA to that of input DNA.

Total protein extracts from NIH3T3 cells were prepared according to the NUN procedure (29 (link)). Antibodies used were rabbit anti-CLOCK 1:3000 (ab3517, abcam), mouse anti-U2AF65 1:5000 (Sigma), rabbit anti-DENR 1:4000 (ab108221, abcam) and anti-rabbit-HRP or anti-mouse-HRP 1:10000 (Promega). Blots were visualized with the Fusion Fx7 system and quantified with ImageJ 1.48k.

Anti-SREBP1 (ab28481), anti-GAPDH (ab8245), anti-CLOCK (ab3517), anti-BMAL1 (ab228594), and anti-SIRT1 (ab110304) were purchased from Abcam (Cambridge, MA, USA). Antibodies against AMPKα (cs5831) and p-AMPKα (cs2535) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against PER2 (AB2202) was purchased from Millipore (Billerica, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Abcam.