Smarcc1

BT12 cells were incubated with 100 nM mithramycin or PBS control for 8 or 18 h. Cells were cross‐linked, lysed, and sheared as described (Harlow et al, 2019). 10 µg solubilized chromatin was immunoprecipitated with 1 µg mouse IgG and 1 µg H3K27me3 (Abcam); 2 µg rabbit IgG and 2 µg SMARCC1 (Cell Signaling); 1 µg rabbit IgG; and 1 µg H3K27ac (Active Motif). Antibody–chromatin complexes were immunoprecipitated and purified as described (Harlow et al, 2019). ChIP DNA was quantified with SYBR green relative to a standard curve generated with chromatin from the respective sample for each primer set. qPCR as described above was performed with the following primer sets (GAPDH, MYT1, SOX2, CCND1, SP1). Antibody information and PCR primer sequences are available in Appendix Table S5.

The following antibodies were utilized: SMARCA2 (Cell Signaling, 11966, dilution 1:2000), SMARCA4 (Abcam, ab110641, dilution 1:1000), PBRM1 (Bethyl Labs, A301-591A, dilution 1:1000), SMARCC1 (Cell Signaling, 11956, dilution 1:1000), HDAC1 (Cell Signaling, 34589, dilution 1:1000), VHL (Cell Signaling, 68547, dilution 1:1000), FLAG (Sigma, F3165, dilution 1:1000), Lamin A/C (Cell Signaling, 4777, dilution 1:1000), α−Tubulin (Sigma, T6074, dilution 1:1000), β-Tubulin (Cell Signaling, 2128, dilution 1:1000), β-Actin (Cell Signaling, 3700, 1:1000 and 4970, dilution 1:1000) and total ubiquitin (Cell Signaling, 3936, dilution 1:1000).