Anti rae1 antibody

ICC analyses were performed, with Abcam’s ICC protocol. An anti-RAE1 antibody (Abcam) was used to detect RAE1 protein. The nucleus was counterstained with DAPI. Images were captured with a 40 × C-Apochromat water immersion lens on a Zeiss LSM 700 Confocal, using Zen 2011 software.

ChIP analysis was performed as previously described⁴² with minor modifications. Chromatin was prepared from stable RAE1-overexpressing and control breast cancer cell lines. Briefly, 1 × 10⁶ cells were cross-linked with 1% formaldehyde for 15 min, followed by the addition of glycine at 125 mM. Chromatin was sheared by sonication to fragments averaging between 0.5 and 1 kb in buffer containing 1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), and protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Chromatin was pre-cleared with protein A/G beads containing 50% slurry (Santa Cruz Biotechnology, Dallas, TX, USA) and salmon sperm DNA, followed by immunoprecipitation with anti-RAE1 antibody (Abcam) coupled to protein A/G beads under each experimental condition. Nonimmune mouse IgG (Santa Cruz Biotechnology) was used as a control. ChIP-PCR data are shown as the percentage of input after normalization with IgG. Primers for ChIP-PCR are listed in Table 2.Table 2

Primer sequences used for ChIP-PCR assay.

Amplicon sites	Sequence (5′ → 3′)
pZEB1 #1	F- GGA TCC CAC GGT TCT ACG C
	R- GCG ACC GGA GAG AGG CTA
pZEB1 #2	F- CTC ATC AAG GGA ACT CCC CG
	R- GAA TTG AGG GGC GAG GGA AA
pZEB1 #3	F- CCC ACC ACA CCT GAG GAA AA
	R- CAT GAT CCT CTC GCT TGT GTC
Gene desert	F- TGG TGG TCT GCC TTC TGC CAG T
	R- TCA CGT GGG AGG AAG AAG TAG GGC