Nb100 77282

The antibodies used along this study are to KDM3A (12835; Proteintech; and NB100-77282; Novus Biologicals, Littleton, CO); KDM3B (IHC 00189; Bethyl Laboratories, Montgomery, TX); Cct4 (ARP34271; Aviva); anti–mono- and dimethylated lysines (ab23366 and ab76118; Abcam, Cambridge, MA); anti-GFP (sc-8334), monoclonal to GAPDH (5019A-2; Imgenex, San Diego, CA), β-gal (A-11132; Molecular Probes), GST (C83271; LSBio); HP1a (clone 15.1952; Upstate); γ-tubulin (GTU-88; Sigma-Aldrich, St. Louis, MO), β-actin (ab8229; Abcam), Hsp90ab1 (MAB32861; R&D Systems), Hsp90aa1 (10713715; Pierce, Rockford, IL), Actbl2 (ab134977; Abcam). Secondary antibodies: anti-mouse and anti-rabbit F(ab′)2 immunoglobulin G Alexa Fluor 488 and 584 (Molecular Probes) for immunofluorescence or horseradish-conjugated peroxidase for immunoblots.

Chromatin immunoprecipitation assays were performed on formaldehyde-treated HCT116 cells essentially as previously described (73 (link)). ETV1 and JMJD1A were precipitated with the rabbit polyclonal antibodies, ab81086 (Abcam) and NB100-77282 (Novus Biologicals), respectively, while control rabbit IgG was purchased from Santa Cruz Biotechnology, Inc. (sc-2027). Approximately 2 µg of the antibodies were employed for the immunoprecipitations that were performed at 4°C over-night (74 (link)). Genomic DNA was amplified in two steps with nested primers (75 (link)). The first PCR encompassed the following temperature steps: 97°C for 1 min; 9 cycles of 97°C for 20 sec, 65°C (-1°C per cycle) for 20 sec, 72°C for 40 sec; 20 cycles of 97°C for 20 sec, 56°C for 20 sec, 72°C for 40 sec; 72°C for 4 min followed by cooling down to 4°C. The second PCR was performed in the same manner except that 31 instead of 20 cycles were used. The primer sequences are presented in Table SII.