Anti phospho h3

The primary antibodies used were as follows: rabbit anti-INPP5E (17797-1-AP; Proteintech); rabbit anti-INPP5E (HPA065758; Sigma), rabbit anti-DDK (14793S; Cell Signaling); rabbit antipericentrin (ab4448; Abcam); mouse anti-PLK1 (ab17056; Abcam); mouse anti-gamma-tubulin (GTU-88; Sigma); mouse phospho-Aurora (2914S; Cell Signaling); mouse anti-alpha-tubulin (A11126; Life Technologies); mouse anti-PI(4,5)P₂ (Z-P045; Echelon Biosciences), rabbit anti-CENPA (2186S; Cell Signaling); mouse antiactin (A5441; Sigma); mouse anti-GAPDH (sc-365062; Santa Cruz Biotechnology), anti-phospho-H3 (9701L; Cell Signaling), and anti-cyclin B1 (4135S; Cell Signaling); and mouse anti-lamin A+C (ab40567; Abcam).

The Western Blot analysis was performed as described previously [31 (link)]. To determine the alterations of protein levels, 40 µg of total protein were separated by 12% and by 7.5% denaturing SDS-PAGE and transferred onto 0.2 µm nitrocellulose membranes overnight. The membranes were blocked with a 5% milk powder solution for 1 h and incubated with primary anti-PARP-IgG (1:1.000, Cell Signaling, #9532), anti-H2AX-IgG (1:10.000, Millipore, #07-627), anti-γH2AX-IgG1 (1:5.000, Millipore, #05-636), anti-cyclin-B1 (1:5.000, Santa Cruz, #sc-752), or anti-phospho-H3 (1:1.000, Cell Signaling, #9701) at 4 °C overnight. The membranes were then incubated with secondary horseradish-peroxidase-coupled antibodies goat-anti-mouse IgG (H+L) (1:10.000, Thermo Fisher Scientific, #A-11029) or goat-anti-rabbit IgG (H+L) (1:10.000, Thermo Fisher Scientific, #31460) for 1 h at rt. GAPDH served as a control for equal loading of the samples, the blots were incubated with HRP-labeled anti-GAPDH (1:100.000, Abnova, #MAB5476) for 45 min at rt. The blots were developed using the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate kit (Merck Millipore). Images were captured using a GeneGnome (Syngene).

2×10⁵/well cells were treated with chidamide in the absence or presence of and/or MI-3 for 48 h, and the subjected to western blot analysis using indicated primary antibodies and secondary HRP-conjugated antibodies (1:10,000, Abcam, Cambridge, UK). The primary antibodies included anti-caspase-3 (#9662S), anti-PARP (#9532S), anti-histone H3 (#4499S), anti-phospho-H3 (#53348S), anti-γH2A.X (#2577S), anti-RAD51 (#8875S), anti-KU70 (#4588S), anti-STAT3 (#9139 S), anti-Mcl-1 (#94296S), anti-phospho-p53 (#9286S), anti-p21(#2947S), anti-phospho-ATM (#5803S), anti-ATM (#2873S), anti-phospho-ATR (#2835S), anti-CHK1 (#2360), anti-CHK2 (#2662), anti-P-CHK1 (#2197S), and anti-P-CHK2 (#2348S) from Cell Signaling Technology (Boston, MA, USA). The primary antibodies were diluted with 5% fat-free milk-TBST. Anti-β-actin (1:1000, Cell Signaling Technology) was used as loading control. Blots were then detected using the ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK).