Ab93694

Histone H3 (WB 1:5000), H3K9me1 (WB 1:1000), H3K9me3 (WB 1:1000) and Mcm3 (WB 1:2000) were generated in the laboratory and were used previously (25 (link),26 (link)). Anti-GFP (ab6556, WB 1:10 000), Polδ (ab186407, WB 1:1000), Polϵ (ab74308, WB 1:1000), KDM4D (ab93694, WB 1:2000, IF 1:200, ChIP 4 μg per IP), Mcm6 (ab201683, ChIP 4 μg per IP), Orc2 (ab68348, WB 1:2000), anti-SLD5 (ab139683, WB 1:1000) and anti-CldU (ab6326) were purchased from Abcam; Orc5 (sc-20635, WB 1:1000), Cdc45 (sc-20685, WB 1:1000), PCNA (sc-56, WB 1:1000) and Tubulin (sc-9104, WB 1:5000) were purchased from Santa Cruz Biotechnology; H3K27me3 (C36B11, WB 1:1000) and H3S10ph (D2C8, WB 1:1000) were purchased from Cell Signaling Technology.
Fluorescein isothiocyanate (FITC)-conjugated anti-BrdU (347583) and anti-IdU (347580) antibodies were purchased from BD Biosciences; FITC goat anti-mouse, FITC goat anti-rabbit and Cy3 goat anti-rat antibodies were purchased from Jackson ImmunoResearch.

Cells were fixed with 3.7% paraformaldehyde at room temperature for 15 min, washed twice with PBS, and permeabilized with 0.3% Triton X-100 (Sigma) for 15 min. After another PBS wash, cells were treated with normal donkey serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) diluted to 5% (v/v) in PBS for 4 hours at room temperature followed by overnight incubation with primary antibodies diluted (1:100, v/v, if not specified) with 1% BSA in PBS at 4°C. Primary antibodies include rabbit monoclonal anti-histone H3 (tri-methyl K9) antibodies (Abcam, ab176916, 1:2000 diluted) and rabbit polyclonal anti-RNA Pol II CTD repeat YSPTSPS (phospho-S2) (Abcam, ab5095). Cells were washed with PBS three times for 5 min each and incubated with the secondary antibody [donkey anti-rabbit immunoglobulin G (IgG) H&L (Heavy & Light chains), Alexa Fluor 555, Abcam, ab150062] diluted with PBS (1:200 v/v) at room temperature for 1 hour. For Western blotting, we used histone H3 (tri-methyl K9) rabbit monoclonal antibody (1:1000, Abcam, ab176916), KDM4D rabbit polyclonal antibody (1:200, Abcam, ab93694), SUV39h1 rabbit monoclonal antibody (1:1000, Cell Signaling, #8729), glyceraldehyde-3-phosphate dehydrogenase rabbit polyclonal antibody (1:1000, Sigma, G9545), and goat anti-rabbit IgG (horseradish peroxidase linked, 1: 1000, Cell Signaling, #7074).