ChIP-PCR Analysis of ELK1 Binding

ChIP-PCR assays were performed as previously described [61 (link)]. In brief, 1 × 10⁷ KYSE30 and KYSE150 cells were crosslinked with formaldehyde (1%) (28908, Thermo Fisher) and neutralized with glycine. Cells were lysed, and DNA was disrupted by sonication (Covaris E220, Woburn, MA, USA). Each ultrasonic product was divided into two equal volumes. Anti-ELK1 and normal IgG were added to each volume and incubated at 4 °C overnight. Dynabeads protein A/G magnetic beads (Invitrogen) were added for 4 h at 4 °C. Complexes were immunoprecipitated, and DNA was eluted and purified using QIAquick PCR Purification Kit (28106, QIAGEN, Germany). The immunoprecipitated DNA was quantified via PCR with SOCS3 promoter-specific primers and separated on a 1.8% agarose gel. The primer sequences used for ChIP-PCR are listed in Supplementary Table S5. The relative enrichment was normalized to a 1% input. IgG antibody was used as a negative control.