Ehmt2 g9a

Whole-cell lysates were prepared from snap frozen cell pellets using RIPA lysis buffer with Complete Mini Protease inhibitors and Phosphatase inhibitors (Roche). Nuclear and Cytoplasmic extracts were prepared from cell pellet or tissue sample using NE-PER™ Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). Equal amount of the whole cell lysates or nuclear/cytosolic extracts were loaded and separated by SDS–PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA), followed by immunoblotting analysis with antibodies to EHMT2/G9a (abcam, ab185050), Di-Methyl-Histone H3 (cell signaling, #4658), Histone H3K9me1 (santa cruz, #39681), Histone H3 (cell signaling, #4499), Tri-Methyl-Histone H3 (cell signaling, #4909), β-actin (Sigma, A2228), phospho-RelB (cell signaling, #5025), RelB (cell signaling, #10544), Bim (cell signaling, #5392), NF-κB2 (cell signaling, #3017), HDAC4 (cell signaling, #5392), HRP-conjugated anti-Rabbit (Cell Signaling #7074), and HRP-conjugated anti-Mouse (Cell Signaling #7076). All primary antibodies were used at 1:2000 dilution and secondary antibody used at 1:10,000 dilution. The blots were developed using ChemiDoc™ MP Imaging System (Bio-rad).