Control igg 2729

A total of 4 × 10⁶ cells were fixed with 1% formaldehyde, lysed, and sonicated (Branson Sonicator; Branson Ultrasonics, Danbury, CT, USA) leading to a DNA average size of 200 bp. 5 µg of antibodies anti-STAT3 #4904 (Cell Signaling), H3K4me3 (ab8580, Abcam, Cambridge, UK) H3K27Ac (ab177178, Abcam), H3K27me3 (ab6002, Abcam), or control IgG #2729 (Cell Signaling) were added to the precleared sample and incubated overnight at 4  °C. The complexes were purified using CHIP-grade protein-G magnetic beads #9006 (Cell Signaling), followed by elution from the beads and reverse cross-linking. DNA was purified using PCR purification columns (Qiagen) and target control genes (TNFRSF8 and GAPDH were amplified by real-time quantitative PCR using SYBR Green (Bio-Rad, #1725272). The oligonucleotide primer pairs are reported in Table S5. Raw ChIP-Seq samples were aligned using bwa aligner. Peak calling was performed using Model-based Analysis of ChIP-Seq (MACS Version 2.1.1, [72 (link)]), an open source computational algorithm for identifying genome-wide protein-DNA interaction from ChIP-Seq data (https://github.com/taoliu/MACS). Only peaks with FDR < 0.05 were called as significantly enriched. Signal tracks were created using MACS2 bdgcmp command. Peak enrichment relative to IgG was calculated in bedgraph format and then converted to bigwig using UCSC toolkit.

Control and higher dose Mortaparib^Plus-treated cells were harvested after 24 h and lysed using the non-ionic NP-40 buffer. The protein concentrations of whole-cell lysates were measured by BCA assay (Thermo Fisher Scientific, Rockford, IL). Cell lysates containing 300–500 μg of total protein were incubated overnight with control IgG (2729) (Cell Signaling Technology) and an antimortalin antibody (raised in our laboratory) in slow rotation at 4 °C. Lysates were centrifuged at 2500 rpm for 3 min, followed by the addition of A/G PLUS-Agarose beads (Santa Cruz Biotech Inc. sc-2003) and incubation of the mixture in slow rotation at 4 °C for 4 h. Immunoprecipitants were collected by cold (4 °C) centrifugation at 2500 rpm for 5 min in a microcentrifuge. Supernatants were removed from the beads and discarded. Pellets containing beads and protein(s) of interest were washed 3–4 times with NP-40 buffer, then centrifugated at 4 °C (2500 rpm) for 5 min. Pellets were mixed with SDS buffer and boiled at 96 °C for 10 min. The immunoprecipitants were resolved on SDS-PAGE, transferred to a PVDF membrane, and then detected with specific antibody for Western blotting.