Millipore chip kit

Chromatin immunoprecipitation (ChIP) was performed using Millipore Chip Kit (catalog #17-10085) and procedures were according to the manufacturer’s protocol and a previously study^{61 (link)}. Shortly speaking, cells cultured under the previously indicated conditions were fixed in 1% formaldehyde/PBS for 10 min at room temperature. After two washes with PBS, cells were resuspended in 0.5 mL of lysis buffer containing a protease inhibitor cocktail before sonication. DNA fragments from the soluble chromatin preparations were 400–800 bp in length. Immunoprecipitation was carried out overnight with purified anti-ELK1(abcam, ab32106), anti-NFκB p65 (abcam, ab19870) or normal rabbit IgG as a negative control. Protein A/G agarose was used to pull down the antigen-antibody compounds and then washed four times with washing buffers. The DNA-protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed using 2 μL DNA samples with the following primers: LRG-1 primer: forward, 5′-TGTCACTACATTTCACAAGCCT-3′; reverse, 5′-CCAGCCGTTAGTTGGTCTTA-3′

ChIP assay was performed using Millipore Chip Kit (#17-10085) according to the manufacturer's protocol. Briefly, cells cultured under the previously indicated conditions were fixed in 1% formaldehyde/PBS for 10 min at room temperature. After two washes with PBS, cells were resuspended in 0.5 mL of lysis buffer containing a protease inhibitor cocktail before sonication. DNA fragments from the soluble chromatin preparations were 400-800 bp in length. Immunoprecipitation was carried out overnight with purified anti-MEF2A antibody (Santa Cruz, sc-17785, 1:200) or anti-NF-κB p65 antibody (Santa Cruz, sc-8008, 1:200) or normal rabbit IgG as a negative control according to a previous study 27 . Protein A/G agarose was used to pull down the antigen-antibody compounds and then washed four times with washing buffers. The DNA-protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed using 2 μL DNA samples with the following primers: Smad7: forward 5′-GAATCTTACGGGAAGATCAAC-3′, reverse 5′-CGCAGAGTCGGCTAAGGT-3′.

The ChIP analysis was performed using the Millipore ChIP kit (Millipore, Billerica, MA, USA), following the manufacturer’s instructions with minor modifications. For each assay, the THP-1 monocytes were inoculated into a 10-cm dish (a total of 5 × 106 cells) and fixed with 1% formaldehyde. Cell pellets were resuspended in SDS lysis buffer containing protease inhibitors (1 mM PMSF, 1 µg/mL aprotinin, and 1 µg/mL pepstatin A). The samples were sonicated with a Misonix sonicator 3000 (Misonix, Farmingdale, NY, USA), centrifuged, and diluted 10-fold in ChIP dilution buffer. After removing an aliquot (whole-cell extract input), the chromatin samples were incubated at 4 °C overnight with antibodies against AP-1 (#9165, Cell Signaling) or NF-κB (#8242, Cell Signaling). The samples were then precipitated by binding to protein A-agarose/salmon sperm DNA beads (Millipore, Billerica, MA, USA). The immunoprecipitated chromatin was analyzed by PCR using primers for the Mac-1 gene promoter. Cycling parameters were 58 °C for 1 min and 95 °C for 30 s, followed by 40 cycles.

Chromatin immunoprecipitation (ChIP) assays were performed using the Millipore ChIP kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions, with minor modifications. Briefly, THP-1 cells were inoculated into a 10-cm dish (5 × 10⁶ cells/dish) and fixed with 1% formaldehyde. Cell pellets were resuspended in SDS lysis buffer containing protease inhibitors (1 mM PMSF, 1 mg/mL aprotinin and 1 mg/mL pepstatin A), and the cell suspension was sonicated using a Misonix sonicator 3000 (Misonix, Farmingdale, NY, USA), centrifuged, and diluted 10-fold in ChIP dilution buffer. After removing an aliquot (whole-cell extract input), the chromatin samples were incubated with antibodies against NF-κB p65 (ab7970 Abcam) or CREB (ab31387) overnight at 4 °C. The samples were then precipitated by binding to protein A-Agarose/Salmon Sperm DNA beads (Millipore, Billerica, MA, USA). The immunoprecipitated chromatin was analyzed using PCR with the primers for the SIRT1 gene promoter and the following cycling parameters: 40 cycles of 58 °C for 60 s and 95 °C for 30 s.