Mab3662

Guide RNAs (sgRNAs, see Appendix Table S2) specific to several different exons of KAP1 (exons 1 and 9) were designed using the website: http://crispr.mit.edu/and cloned into the PX459 plasmid (Addgene), which was then transfected into HeLa and HEK293 T cells. After 24 h, the cells were subjected to puromycin selection for 24 h or until control cells had completely died. The bulk population was then used for single‐cell cloning by limiting dilution. Knockout was assessed across a panel of clones by Western blotting using the KAP1 antibody, MAB3662 (Millipore), and validated functionally using KAP1‐KZNF reporter assays. For knockout of STING or MAVS, sgRNAs (listed in Appendix Table S2) were cloned into lentiCRISPRv2 and THP‐1 cells transduced, selected with puromycin and single cell cloned as above.

Antibodies used in these studies include: mouse anti-TRIM28 (MAB3662, Millipore), mouse anti-PCNA (NA03, Calbiochem), mouse anti-HP1Y (05-690, Millipore).

Cells were washed with ice-cold PBS and resuspended in radioimmunoprecipitation (RIPA) buffer to prepare total cell extracts. Protein amount was quantified by BCA protein assay reagents (Pierce) and normalized for loading on a 10% denaturing SDS–polyacrylamide gel. Wet transfer was performed, and the primary antibodies used were anti-Trim28 (mouse mAb; MAB3662 Millipore), anti-ZFP57 (ab45341, Abcam) and anti-beta Actin HRP (ab20272, Abcam).