O glcnac rl2

For western blot experiments, ChIP grade mH2A1 (Abcam, ab37264), GAPDH (Abcam, ab9484), H2AK119Ub (Millipore, #AB10029) and O-GlcNAc (RL2) (Thermoscientific, MA1-072) antibodies were used at a dilution of 1:1000. For immunofluorescence experiments, O-GlcNAc (RL2) (Thermoscientific, MA1-072), OGT (TI14) (Sigma Aldrich, O6014), RNA polymerase II (phospho S2) (Abcam, ab5095 and ab5408), RNA polymerase II (phospho S2/5) (Cell signaling, #4735), Actin (Abcam, ab1801) and H3K27Me3 (Millipore, #07-449) antibodies were used at a dilution of 1:200, mH2A1 (Cell signaling, #4827) and pan RNA polymerase II (RBP8) (Abcam, ab169924) at a dilution of 1:100.

Cells were homogenized in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40) and protease inhibitor cocktail (Sigma). Total proteins (10–20 μg) were separated by SDS-PAGE on 10% acrylamide gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies: O-GlcNAc (RL2) (1:10,000; Thermo Fisher Scientific Inc., Rockford, IL, USA), OGT (1:5,000; Santa Cruz Biotechnology, CA, USA), AMPKα (1:5,000), p-AMPKα (1:5,000; Cell Signaling), p21 (1:5,000; Santa Cruz Biotechnology), p27 (1:5,000; Santa Cruz Biotechnology), poly(ADP-ribose) polymerase (PARP, 1:5,000; Cell Signaling #9532), cleaved PARP (1:5,000; Cell Signaling #5625), and β-actin (1:10,000; PIERCE, Rockford, IL, USA). Immunoreactive antigens were detected using the Enhanced Chemiluminescence Detection kit (ECL, Amersham Bioscience, Piscataway, NJ, USA). The specific protein bands were analyzed by densitometry, and the values were normalized to that of the β-actin control and further analyzed using Image J software (Bethesda, MD, USA).