Ab10483

HEK293 cells (5 × 10⁶) were transfected with 10 μg pCMV2B-ER and 0.5 μg pCR2.1-MCPyV-NCCR or 10 μg pCMV2B-ER and 0.5 μg pMK-MCVSyn-stop. At 2 days p.t., ChIP was performed as described previously (57 ) using anti-LT antibody (Cm2B4) and anti-Kap1 antibody (ab10483; Abcam).
Sequencing libraries were prepared from 2 to 10 ng DNA using a NEXTflex Illumina ChIP-seq library prep kit (Bio Scientific) and were sequenced on an Illumina NextSeq (SR50) system. Sequencing reads were aligned to the human reference genome (hg19) and pCR2.1-MCPyV-NCCR using Bowtie version 1.2.2 (58 (link)). Sites with enriched Kap1 levels were detected by the use of MACS version 2.1.2 (59 (link)), and matched negative-control region sets were generated with EaSeq (60 (link)).

For knockdown experiments, 4 × 10⁶ N2A cells per ChIP were seeded in 15‐cm plates. The next day, cells were transfected with either 15 μg Paupar targeting shRNA expression vectors or a non‐targeting scr control. Three days later, cells were harvested for ChIP using either 5 μg anti‐KAP1 (ab10483, Abcam), anti‐histone H3K9me3 (39161, Active Motif) or normal rabbit control IgG (#2729, Cell Signalling Technology) antibodies. ChIP was performed as described in Vance et al (2014). 5 μg anti‐PAX6 (#AB2237, Millipore) was used for PAX6 ChIP. For KAP1 ChIP‐seq, the following modifications were made to the protocol: ~2 × 10⁷ N2A cells per ChIP were double‐cross‐linked, first using 2 mM disuccinimidyl glutarate (DSG) for 45 min at room temperature, followed by 1% formaldehyde for 15 min at room temperature, as described in Nowak et al (2005). Chromatin was sheared to ~200 bp using a Bioruptor Pico (Diagenode) and ChIP DNA and matched input DNA from two independent KAP1 ChIP experiments were sequenced on an Illumina HiSeq 4000 (150‐bp paired‐end sequencing).

The list of antibodies we used was as follows: anti-SETDB1 (Upstate 07-378 for NT and Abcam ab12317 for CT), -DICER1 (sc-30226, Santa Cruz), -AGO2 (2897, Cell signaling), -AR (3202, Cell signaling), -PR (3176, Cell signaling), -β-actin (sc-47778, Santa Cruz), -KAP1 (ab10483, Abcam), -EZH2 (3147, Cell signaling), -SIN3A (ab129087, Abcam), -HDAC1 (sc-7872, Santa Cruz), -HDAC2 (sc-7899, Santa Cruz), -MTA2 (ab8106, Abcam), -DNMT3A (D23G1, Cell signaling) and -DNMT3B (ab13604, Abcam).

Whole-cell lysates were prepared by lysing the cells with radioimmune precipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) completed with Complete Protease Inhibitor Mixture (Roche) according to the manufacturer’s protocol. Protein lysates were stored in −80 °C for further analyses. Protein concentration was measured with PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and then samples were subjected to SDS-PAGE (using Mini-PROTEAN^® Tetra Cell System, Bio-Rad) followed by immunoblotting with antibodies for TRIM28 (ab10483 or ab10484, Abcam, Cambridge, UK) and GAPDH (ab9485, Abcam). The blots were visualized using an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Biosciences, Little Chalfont, UK) and a G:BOX F3 Gel Documentation System (Syngene, Bangalore, India).

1 × 10⁶ N2A cells were seeded per 10‐cm dish. The next day, cells were transfected with different combinations of pcDNA3‐FLAG‐PAX6, pcDNA3‐HA‐KAP1, pcDNA3‐RCOR3, pCAGGS‐Paupar, pCAGGS‐AK034351 control transcript or pcDNA3.1 empty vector. 6 μg plasmid DNA was transfected in total. Two days later, cells were washed twice with ice‐cold PBS, transferred to 1.5‐ml microcentrifuge tubes and lysed in 1 ml ice‐cold IP Buffer (IPB; 50 mM Hepes pH 7.5, 350 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA and 0.4% IGEPAL CA‐630) for 30 min, 4°C with rotation. Lysates were pelleted at 16,000 g, 20 min, 4°C in a microfuge, supernatant was added to 30 μl anti‐FLAG M2 Magnetic Beads (#M8823, Sigma) and incubated overnight at 4°C with rotation. Beads were washed three times with IPB and eluted in 20 μl Laemmli sample buffer for 5 min at 95°C. Bound proteins were detected by Western blotting using anti‐FLAG M2 (F3165, Sigma), anti‐KAP1 (ab10483, Abcam), anti‐RCOR3 (A301‐273A, Bethyl Laboratories) and Protein A HRP (ab7456, Abcam).