Sc 1081x

ChIP assays were carried out using the ChIP-IT Express Enzymatic kit (Active Motif) using a dounce homoginizer to lyse cells. Optimal enzymatic digestion of chromatin from MDA-MB-231 cells was empirically determined to occur after 10 min, yielding sheared chromatin that migrated between 200 and 1500 bp on an agarose gel. Equal DNA concentrations corresponding to 1.5 μg were applied to each set of immunoprecipitation reactions, which included either normal rabbit IgG, STAT3, or STAT5A antibody (sc-2027, sc-7179X, or sc-1081X, respectively; Santa Cruz Biotechnology). Samples were incubated with magnetic beads overnight at 4°C with end-over-end rotation. After reversal of cross-links, DNA precipitation, and clean-up, enriched DNA and input were analyzed by quantitative real time PCR with primers spanning the predicted GAS site, as well as primers specific to a region of the LKB1 promoter that does not contain a putative STAT binding motif (Table 
2). The efficiency of each primer set was tested by producing a standard curve from two-fold dilutions of input, and the integrity of products was verified by agarose gel electrophoresis. Fold enrichment relative to IgG was calculated for immunoprecipitated samples, and data are presented normalized to values obtained for the negative binding region.

To detect SUMOylated proteins, cell extracts were prepared using lysis buffer supplemented with protease inhibitors plus N-ethylmaleimide (Pierce, Thermo Fisher Scientific) following the detailed protocol of Park-Sarge and Sarge (2009) [33 (link)]. Immunoprecipitation reactions (100 μg of total protein/1 reaction) were carried out with either normal rabbit IgG (DA1E; #3900, Cell Signaling Technology) as a negative control or total STAT5 antibody (sc-1081X, Santa Cruz Biotechnology). Immunocomplexes were isolated using protein A magnetic beads (#8687, Cell Signaling Technology) followed by standard Western blotting with either total STAT5, SUMO-1 (C9H1; #4940, Cell Signaling Technology), SUMO-2/3 (18H8; #4971, Cell Signaling Technology), or calnexin antibody as a negative control. Non-immunoprecipitated input was run in parallel on the same blots as a positive control.