Ab9262

Proteins were harvested from each treatment condition as described previously[18 (link)]. Membranes were blotted with rabbit anti-SMC1A primary antibody (Abcam; ab9262; [1:10,000]). Blots were stripped and re-blotted with mouse monoclonal anti-α-tubulin antibody (Abcam; ab7291; [1:4000]) as a loading control. All primary antibodies were visualized by secondary antibodies conjugated to horseradish peroxidase. Blots were imaged and bands were visualized on a MyECL Imager (Thermo Scientific) using standard chemiluminescence.

Primary antibodies used in this study were anti-RAD21 1:500 (Abcam, ab992), anti-SMC1A 1:500 (Abcam, ab9262), anti-SMC3 1:500 (Abcam, ab9263), anti-STAG1 1:500 (Bethyl, A302-579A), anti-STAG2 1:200 (Santa Cruz, sc-81852), anti-histone H3 1:1000 (Abcam, ab1791), anti-MEK2 1:500 (BD, 610235), anti-nucleolin 1:500 (Santa Cruz, sc-8031), anti-AcSMC3 1:500 (MBL, PD040), and anti-NIPBL 1:500 (KT55, Abcam, ab106768) for immunoblotting and 5 µl anti-NIPBL (3B9, Novus Biological H00025836-M01), 25 µl H-300 (Santa Cruz, sc-98601), and 25 µl C-9 (Santa Cruz, sc-374625) for immunoprecipitation (Supplementary Fig. 5). Custom-made rabbit polyclonal antibodies against PDS5A (1:500) have been described^{89 (link)} and those for human sororin (1:500) were raised using the recombinant protein purified from bacteria as antigen. Horseradish peroxidase HRP-conjugated (1:10,000) secondary antibodies (Amersham Biosciences) were used. Immunoblots were quantified using ImageJ software (Fiji v1.53).

Genomic DNA was treated using bisulfite, amplified (primers in Table S2), and subcloned. DNA from clones (>10 clones/amplicon) was sequenced as described elsewhere [15] (link).
For co-IP, 6×10⁶ MEL or K562 cells were lysed (RIPA) and gently sonicated. 100 µg of precleared protein extract was incubated 3 hrs/4°C with anti-Ctcf (cat#07–729) or anti-Smarca5 (cat#07–624, Upstate) and next with proteinA/proteinG overnight. Control antibody: IgG, cat.# NI01, Calbiochem, 5∶100). Immunoprecipitates (IP) were washed with set of buffers. IPbuffer (0.02% SDS/2%Trion X-100/4 mM EDTA/40 mM Tris-HCl (pH = 8)/300 mM NaCl), WashI (0.1%SDS/1%Triton X-10/2 mM EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/50 mM NaCl), WashII (0.1% SDS/1% Triton X-10/1% EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/500 mM NaCl). IPs were resolved on SDS/PAGE, blotted, and immune-detected.
Chromatin immunoprecipitation (ChIP) [19] (link) lysates were controlled for DNA purity&quantity by Nanodrop ND-1000. Antibodies: Smarca5/Snf2h (cat.#ab3749, Abcam, 3 µg/IP), Ctcf (cat.#ab10571, Abcam, 2 µg/IP), RAD21 (cat.#ab992, Abcam, 2 µg/IP), and SMC1 (cat.# ab9262, Abcam, 2 µg/IP). Control IgG: cat.#NI01, Calbiochem, 5∶100.