Ab194286

ChIP-quantitative polymerase chain reaction (qPCR) was conducted following the standard protocol of commercial kits (Beyotime, China). Briefly, hPDLSCs were fixed with 1% formaldehyde and the cell lysis buffer was processed by ultrasound to obtain fragments. The fragments were incubated with the primary antibody KDM5A (ab194286, Abcam) or immunoglobulin G (ab133470 Abcam) at 4 °C overnight to conduct Immunoprecipitation reaction. Then, the precipitate was eluted and immunoprecipitated DNA was purified by universal DNA purification kits (TIANGEN BIOTECH Co., Ltd., Beijing, China). Purified DNA was analyzed using real time-qPCR (RT-qPCR). The enrichment level was calculated according to a previous method [22 (link)]. First, two ChIP DNA fractions were normalized to input DNA fraction, where input dilution factor was 10 as 10% of IP reaction was used as input according to the formula: ΔCt [normalized ChIP] = Ct [ChIP] − (Ct [input] Log₂ input dilution factor). Meanwhile, ΔCt [normalized IgG mock] was calculated for IgG (mock) sample. Lastly, the fold enrichment was calculated as 2 ^{−[ΔCt normalized ChIP − ΔCt normalized mock]}. ChIP primers: forward primer, 5′-GATGTGTTTCTCACGGTTTGCC-3′; reverse primer, 5′-CACGGTAATTGTGCATCATTTG-3′.

The cells were lysed using high RIPA lysis buffer (P1003B, Beyotime, Shanghai, China) to obtain total protein. Bicinchoninic acid (BCA) kits (Beyotime) were used to detect protein concentration. Proteins were separated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Kaihong, Shenzhen, Guangdong China). The membrane blockade was conducted using Tris-buffered saline tween buffer containing 5% skim milk at 37 °C for 1 h, followed by incubation with the primary antibodies Runt-related transcription factor 2 (Runx2) (1:1000, ab236639, Abcam), osteocalcin (OCN) (1:1000, ab133612, Abcam), osteopontin (OPN) (1:1000, ab214050, Abcam), KDM5A (1:5000, ab194286, Abcam), H3K4me3 (1:1000, ab213224, Abcam) and β-actin (1:100, ab8227, Abcam). After overnight incubation with the primary antibody, the membranes were cultured with the secondary antibody (1:2000, ab6721, Abcam) at room temperature for 2 h. Finally, enhanced chemiluminescence (Thermo Fisher, Waltham, MA, USA) was used to observe Western blot, and Image J software (NIH, Bethesda, MD, USA) was used to analyze the relative expression of proteins with β-actin as the internal reference.