TCL and NCF, and histones were resolved on 10 and 18 % polyacrylamide SDS-PAGE, respectively, and transferred to PVDF membrane. Proteins on PVDF membrane were hybridized with anti-H3 (Upstate-06-755; 1:2000 dilution), H4 (Millipore-07-108; 1:4000 dilution),
H3S10ph (Millipore-06-570; 1:7000 dilution),
H4K16ac (Millipore-07-329; 1:8000 dilution),
H4K20me3 (Abcam-9053; 1:4000 dilution),
β-actin (Sigma-A5316; 1:10,000 dilution), MSK1 (Santacruz-9392; 1:2000 dilution),
ph-MSK1 (Abcam-31190; 1:3000 dilution),
ERK1/2 (Santacruz-292838; 1:2000 dilution),
ph-ERK (Cell signaling-9910; 1:2000 dilution), p38 (Santacruz-728; 1:2000 dilution),
ph-p38 (Cell signaling-9910; 1:2000 dilution), and
anti-flag (Sigma-F3165; 1:5000 dilution). Signal was visualized using horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibody and
ECL plus chemiluminescence kit (Amersham). Wherever required, the densitometry analysis was done on immunoblot and membrane to determine their mean intensities using ImageJ software. For native proteins, mean intensity of immunoblot was normalized with the stained PVDF membrane; for phosphorylated forms, mean intensity of immunoblot was normalized with immunoblot of native proteins. The resulted value was used to express their mean relative levels in resection margin and tumor.
Khan S.A., Amnekar R., Khade B., Barreto S.G., Ramadwar M., Shrikhande S.V, & Gupta S. (2016). p38-MAPK/MSK1-mediated overexpression of histone H3 serine 10 phosphorylation defines distance-dependent prognostic value of negative resection margin in gastric cancer. Clinical Epigenetics, 8(1), 88.