Anti bmal1 d2l7g

After animals were killed, draining LNs were snap frozen on dry ice and stored at −80 °C before processing. Tissue was dounce homogenized in 1 ml of homogenization buffer (10 mM HEPES-KOH, 10 mM KCl, 5 mM MgCl₂, 0.5 mM DTT and 1× Complete Protease Inhibitor (Roche)). The homogenate was fixed in PBS containing 1% final formaldehyde (Thermo Fisher Scientific). Nuclei were isolated, and chromatin suspensions were obtained through sonication (Diagenode Bioruptor) to obtain fragments of 0.2–0.8 kilobases in size. Immunoprecipitation was performed with anti-BMAL1 (D2L7G, Cell Signaling Technology) or control IgG (Abcam). DNA was isolated with MinElute PCR Purification kits (Qiagen). DNA concentration was determined by using a Qubit dsDNA HS kit (Thermo Fisher Scientific). Real-time ChIP–qPCR was performed by use of SYBR Green Master Mix (Roche) in a LightCycler 480 II (Roche). Occupancy of BMAL1 at the Ccr7, Ccl21, Lyve1 and Per2 promoters was quantified by qPCR targeting regions identified as containing E-boxes using the SCOPE motif finder and EPFL eukaryotic database. Relative enrichment was determined as percentage of the input.

A total of 2 × 10⁷ BMDCs were collected and fixed in PBS containing 1% formaldehyde (Thermo Fisher Scientific) for 10 min at room temperature and quenched with 1 M glycine in PBS. Cells were then pelleted and sonicated (Diagenode Bioruptor) to obtain fragments of 0.2–0.8 kilobases in size. Immunoprecipitation was performed using anti-BMAL1 (D2L7G, Cell Signaling Technology), anti-histone H3 (Abcam) or control IgG (Cell Signaling Technology). DNA was isolated using the MinElute PCR Purification kits (Qiagen). qPCR was performed using PowerUp SYBR Green (Applied Biosystems) in the StepOne Real-Time PCR System. Occupancy of BMAL1 at the Cd80 and Per2 promoters was quantified by qPCR targeting regions identified as containing E-boxes using the SCOPE motif finder and EPFL eukaryotic database. Relative enrichment was determined as the percentage of input.