Mouse monoclonal anti dnmt1

ChIP assays were performed using a Simple Enzymatic ChIP assay kit (Cell Signaling Technology), according to the manufacturer’s protocol. ChIP assays were performed using the following antibodies: mouse monoclonal anti-DNMT1 (Abcam, no. 92453); mouse monoclonal anti-DNMT3A (Abcam, no. 113430); goat polyclonal anti-DNMT3B (Abcam, no. 13604); rabbit polyclonal anti-USF1 (Santa Cruz Biotechnology, no. sc-229); and rabbit polyclonal anti-SREBF1 (R&D Systems, no. NB100-60545SS).

GBM tissues or cells were lysated in a modified RIPA buffer (50 mM Tris (pH 7.4), 1% Triton X‐100, 150 mM NaCl, 2 mM EDTA, protease inhibitor cocktail, 1 mM β‐glycerophosphate, 1 mM Na₃VO₄, 1 mM NaF). The lysates were centrifuged and cleared by incubation with 25 µl of Protein G‐Agarose (Roche) for 1.5 h at 4 °C. The pre‐cleared supernatant was subjected to IP using the indicated first antibodies at 4 °C overnight. Antibodies used in IP included: mouse monoclonal anti‐CD133 (W6B3C1 clone) (Miltenyi Biotec, cat# 130‐092‐395) and mouse monoclonal anti‐DNMT1 (Abcam, cat# ab13537). Then, the protein complexes were collected by incubation with 30 µl of Protein G‐Agarose (Roche) for 2 h at 4 °C. The collected protein complexes were washed four times with IP buffer and analyzed by western blotting using indicated antibodies. Antibodies included: rabbit polyclonal anti‐DNMT1 antibody (Abcam, cat# ab13537), mouse monoclonal anti‐CD133 (W6B3C1 clone) (Miltenyi Biotec, cat# 130‐092‐395; 1:1000).