Smyd3

Macrophages were lysed in SDS lysis buffer [2% SDS (w/v)/16% glycerol (v/v) in 50 mM Tris, pH 6.8]. Protein was measured (Micro BCA kit, Thermo Scientific Pierce). Equal amounts of reduced protein were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes (Merck Millipore), blocked in TBST/5% skimmed milk followed by incubation in primary antibody. Proteins were detected using appropriate HRP-conjugated secondary antibodies (SigmaAldrich) and enhanced chemiluminescence (Immobilon, Merck Millipore) and Hyperfilm™ ECL (GE Healthcare Life Sciences). The antibodies used were Phospho-Rb-Ser807/811, E2F1, KDM6A, and SMYD3 (Cell Signaling), Histone H3 and Histone H3-Phospho S10 (Abcam), GAPDH (Millipore), and p27^kip1 (BD Biosciences).

The rabbit polyclonal anti-cGAS K491me antibody was produced by Boer Biotechnology (Chengdu, China). The mouse cGAS K491me peptide was synthesized and injected into rabbits. An affinity column with bound nonmodified peptide was used to collect and purify rabbit serum, excluding antibodies targeting nonmethylated cGAS. The antibody was then purified using an affinity column linked with bound cGAS K491me peptide. A rabbit polyclonal antibody recognizing human cGAS K506me was produced following the same protocol.
Antibodies recognizing cGAS, Tubulin, GST, His, SET1A, SET1B, SET7, MLL1, MLL3, γH2A.X and SMYD3 were obtained from Cell Signaling Technology. Antibodies recognizing RIOX2 were purchased from Atlas Antibodies. Antibodies recognizing RIOX1, ASH1L, PARP1, MLL2, SGF29, Timeless, NSD1, NSD2, H2A.X, CD11b, CD11a, CD34, Thy-1, and SMYD2 were purchased from Abcam. The antibody against Flag, anti-Flag M2 agarose beads, streptavidin-conjugated agarose beads, biotin, bovine serum albumin and poly(ADP-ribose) glycohydrolase (PARG) recombinant protein were purchased from Sigma. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific. biotin (terminal)-PAR Polymer was purchased from Trevigen.