Ecl substrate

After culturing in 6-well plates and 21-day induction using an osteogenic medium, we rinsed MC3T3-E1 cells twice with PBS, followed by lysis with RIPA lysis buffer (Solarbio, Beijing, China) supplemented with 1 mM PMSF (Sigma-Aldrich, Shanghai, China) to inhibit protein degradation [18 (link)]. We detected the total protein concentration after centrifugation using a BCA kit (CWBIO, Beijing, China). For the WB assay, we separated proteins (20 μg) using 12% SDS-PAGE, followed by transfer onto PVDF membranes. Thereafter, the membranes were blocked using 5% nonfat milk at 4°C overnight and rinsed three times with TBST. Next, we used primary antibodies (Abcam, Cambridge, UK) to incubate the PVDF membranes for 2 h, including Runx2 (1:1500), OCN (1:1000), Col1A1 (1:2500), ALP (1:1000), and GAPDH (1:3000). The primary antibodies against Smad proteins, including Smad7 (1: 1500), Smad2 (1:2000), Smad3 (1:1000), phospho-Smad2 (1:1500), and phospho-Smad3 (1:2000), were obtained from Beyotime (Shanghai, China). After rinsing thrice with TBST, the membranes were incubated with HPR-labeled secondary antibody (1:6000; CWBIO, Beijing, China). ECL substrate (CWBIO, Beijing, China) and ChampChemi Professional (Sage, Beijing, China) were used to visualize the bands on the membranes. ImageJ software was used to quantify protein levels through densitometry, with GAPDH as the endogenous control.

Genomic DNA (gDNA) of P. capsici was extracted using a TIANGEN DNAsecure Plant kit (Beijing, China) and RNA was removed by RNase treatment and column chromatogram. Equal amounts of gDNA were denatured at 95°C for 5 min and chilled on ice for 10 min. DNA were spotted onto Amersham Hybond-N⁺ membranes (GE Healthcare, Beijing, China), and further dried at 37°C for 30 min. DNA was then crosslinked under UV for 5 min. The membrane was blocked in 5% milk phosphate-buffered saline with Tween 20 (PBST) for 1 h and incubated with 6mA antibody (sysy, 202003) or 5mC antibody (Abcam, ab73938) in 5% milk PBST overnight at 4°C. After a PBST wash, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Proteintech, Beijing, China) for 1 h and treated with ECL substrate (CWBIO, Jiangsu, China). After washing, the signal was detected by Tanon 5200 (Shanghai, China). For input quantification, the same membrane was incubated with 0.1% methylene blue solution for 15 min and washed using tris-buffered saline with Tween 20 (TBST) buffer three times. Relative 6mA abundance was quantified (integrated signal density anti-6mA/integrated signal density input DNA) using ImageJ (NIH image to ImageJ: 25 years of image analysis.).