Anti phospho histone h3 phh3

Total protein was obtained by RIPA lysis buffer (Beyotime) with protease inhibitor cocktail (Thermo Fisher), and protein concentrations were measured by Pierce BCA Protein Assay Kit (Thermo Fisher). A total of 30‐40 ng protein extracts were loaded onto SDS‐PAGE gels (6%‐12%) and then transferred to PVDF membranes. PVDF membranes were blocked by 5% w/v non‐fat dry milk which was dissolved in tris‐buffered saline solution containing 0.1% tween‐20 (TBS‐T) for 2 hours. The PVDF membranes were then incubated with the primary antibodies at 4°C overnight: anti‐NOTCH2 (1:4000 dilution; Cell Signaling Technology), anti‐phospho‐Histone H3 (PHH3) (1:2000 dilution; Cell Signaling Technology), anti‐PCNA (1:2000 dilution; Cell Signaling Technology) and anti‐β‐actin (1:4000 dilution; Cell Signaling Technology). After three washing with TBS‐T, the PVDF membranes were incubated with HRP‐labelled goat anti‐rabbit IgG secondary antibody (1:8000 dilution; Abcam) for 1 hour at room temperature. After the final washing with TBS‐T, protein bands were visualized with ECL (Merck Millipore) reagents.

Tumor tissues were isolated and frozen in freezing medium using ice-cold methyl butane. Tissues were cut in 6 µm sections using a CryoTome (Fisher Scientific, Loughborough, UK); sections were placed on glass slides, then stored at −80 °C or immediately used for staining. Tissue sections were then fixed with 2% paraformaldehyde (PFA) followed by permeabilization with 0.5% Triton X-100. After blocking with 7% horse serum (Vector Laboratories, Burlingame, CA, USA) in PBS, primary antibodies were then applied overnight at 4 °C. Sections were then incubated with fluorescently labeled secondary antibodies for 1 h at room temperature. The images were captured using a Fluorescence Microscope (Zeiss, Thornwood, NY, USA). The following antibodies were used: anti-CD8 (BioLegend), anti-PD-L1 (BioLegend), anti-Phospho-Histone H3 (P-HH3) (Cell Signaling) and anti-CD31 (BD Biosciences).