Mabe191

For whole cell lysates, cells were washed with cold PBS and lysed in RIPA lysis buffer. Extracted histones or whole cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, blocked in 5% non-fat milk in PBS plus 0.1% Tween-20, probed with primary antibodies, and detected with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch). Primary antibodies used include: anti-H3 (CST, 9715S); anti-H3K36me2 (CST, 2901S); anti-H3K36me3 (Abcam, ab9050); anti-H3K36M (Millipore, ABE1447); anti-Nsd2 (Millipore, MABE191); anti-Kdm2a (Abcam, ab191387); anti-alpha tubulin (CST, 3873S); anti-E-cadherin (Takara, M108).

Fractionated or whole cell lysates were resolved by SDS-PAGE, transferred to a nitrocellulose or PVDF membrane, blocked in 5% non-fat milk in PBS plus 0.5% Tween-20, probed with primary antibodies, and detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (GE Healthcare). Primary antibodies were: anti-H3K36me2 (Cell Signaling Tech, 2901), anti-H3K36me3 (Active Motif, 61101), anti-NSD1 (Abbexa, abx135901), anti-NSD2 (Millipore Sigma, MABE191), anti-SETD2 (Abcam, ab31358), anti-DNMT3A (Abcam, ab188470), anti-Vinculin (Cell Signaling Tech, 13901), anti-His (ZSGB-Bio,TA-02), anti-Lamin B1 (Cell Signaling Tech, 12586), anti-β-Tubulin (Cell Signaling Tech, 2128), anti-β-actin (Abcam, ab8224), anti-H3 (Abcam, ab1791) and anti-HA (Biolegend, 901501). The specificities of anti-H3K36me3 and anti-H3K36me2 antibodies were validated using Setd2 knockout and Nsd1/2 double knockout cell lines.