Nonimmune rabbit igg

ChIP assay was performed using ChIP Kit (Bes5001, BersinBio, Guangzhou, China) according to manufacturer’s instructions. Briefly, the cells were fixed with 1% formaldehyde, and the cross-linking was quenched by adding in 100 μl of 1.375 M glycine per milliliter of culture. The samples were sonicated on ice to shear the DNA into 200 to 600 bp fragments. For each total cell lysate, one third was used as the DNA input control, another third was immunoprecipitated with anti-RIF1 antibodies, and the last third was subjected to non-immune rabbit IgG (Cell Signaling Technology). DNA fragments were purified by spin columns (Qiagen, Hilden, Germany), and Real Time PCR was performed to amplify the segment in the promoter region of hTERT with the following primers:
Prime 1, Forward: 5’-TCAAGTCACACCCACTGGTAAG-3′, Reverse: 5’-ATGGGATAACAGGTGGTCACAG-3′. Prime 2, Forward: 5’-ACTGCTGGTACTGAATCCACTG-3′, Reverse: 5’-GCCTGTAATCCCAGCCAAATG-3′. Prime 3, Forward: 5’-GTTTCCTCGCCATGCACATG-3′, Reverse: 5’-GGGGCGGTTTGGAAAATTTG-3′. Prime 4, Forward: 5’-ATTTCCTCCGGCAGTTTCTG-3′, Reverse: 5’-TTGGATCTAAGGGGCGAGAAAC-3′. Prime 5, Forward: 5’-TCCATTTCCCACCCTTTCTCG-3′, Reverse: 5’-AGGCCCGTCATTTCTCTTTG-3′.

Enrichment of C/EBPβ binding at the EDIL3 promotor region was evaluated in hPDLCs treated with or without macrolides and assessed using the SimpleChIP Plus Enzymatic Chromatin IP Kit with magnetic beads (Cell Signaling Technology) as described in previous studies.⁴³ (link)^,99 (link) Briefly, cross-linked chromatin was immunoprecipitated with nonimmune rabbit IgG (Cell Signaling Technology) or anti–histone H3 rabbit IgG monoclonal antibody (mAb) (D2B12; Cell Signaling Technology) or anti-C/EBPβ mouse IgG (H-7; Santa Cruz Biotechnology, Dallas, Texas, USA). For RT-PCR, primers 5′ CTTATAGCAGAAG- GAGCTGAAAGAG 3′ and 5′ TGGAGAACAATGAAGGCGTGAG 3′ flanking 2 putative C/EBPβbinding sites in the EDIL3 promoter (328 to 589 base pairs) were used. ChIP-qPCR data obtained using the anti-C/EBPβ antibody or nonimmune IgG were normalized using a percentage input method that normalizes chromatin input based on the following equation: % input = 100 × 2^{Ct[adjusted input] – Ct[IP]}.⁹⁹ (link)^,100 (link)