Protein a and g sepharose beads

P53/ROSA26-Zeb2^tg/tg primary mouse T-ALL cells were cross-linked in 1% formaldehyde for 10 min at room temperature and stopped with glycine (final concentration 0.125 M). Cells were lysed in the presence of protease inhibitors and DNA was sonicated. For immunoprecipitation, 100 μg DNA was used together with 3 μl of anti-ZEB2 monoclonal Ab49 (link). Complexes were precipitated with protein A and G Sepharose beads (GE Healthcare). Formaldehyde cross-links were reversed by overnight incubation at 65 °C and DNA was purified using QIAquick PCR Purification Kit (Qiagen). Primers used are given in Supplementary Table 6, including localization of the respective amplicon on the mouse Il7r promotor (Supplementary Fig. 10c). Figure was generated using USCS genome bioinformatics software (http://genome.uscs.edu/).

Immunoprecipitation and immunoblotting were performed as described previously 6, 20. In brief, transfected cells were solubilized in ice‐cold IP buffer [20 mm Tris–HCl, pH 7.4, 150 mm NaCl, 10 mm Na₂HPO₄, 1% Triton X‐100, 0.5% Na‐deoxycholate, 0.1% SDS, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride (PMSF)]. Where indicated, 2 mm EGTA or 2 mm CaCl₂ was added in lieu of EDTA. Solubilized lysates were incubated for 16 h at 4 °C with protein A and G sepharose beads (GE Healthcare Biosciences, Marlborough, MA, USA) precoated with the indicated rabbit and mouse antibodies, respectively. Protein samples were separated on 7.5–15% SDS/PAGE, transferred to nitrocellulose membranes, followed by immunoblotting. For detecting centrin signal, membranes were fixed with 0.2% glutaraldehyde prior to primary antibody incubation. Input represents 5% of the total protein used for immunoprecipitation. The antibodies include mouse anti‐β‐actin (Sigma, St. Louis, MO, USA), mouse anti‐centrin (Millipore, Billerica, MA , USA), rabbit anti‐rEag1 (Alomone, Jerusalem, Israel), rabbit anti‐GFP (Abcam, Eugene, OR, USA), mouse anti‐GST (Sigma), mouse IgG (Sigma), mouse anti‐Myc (clone 9E10), mouse anti‐PSD‐95 (NeuroMab, Davis, CA, USA), and mouse anti‐synaptophysin. Results shown are representative of at least three independent experiments.

Cell extracts from a nearly confluent T25 flask were lysed in buffer (see above) and incubated with 1 µg of either anti-CDK2 antibody (BD Biosciences, 610145) or anti-GFP antibody (DSHB, 12A6; used as a negative control) overnight. Antibody–antigen complexes were precipitated using protein A and G sepharose beads (GE Healthcare). Bead complexes were washed three times with lysis buffer. Kinase assay reactions were incubated at 30 °C for 30 min in buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl₂, 0.1 mM NaF, 10 μM Na₃VO₄) with 1 mM DTT, 1 μM cold ATP, 2 μCi [γ-³²P] ATP, and 4 μg histone H1 (Sigma, H1917). Reactions were resolved by SDS-PAGE. Dried gels were exposed to a storage phosphor screen (GE Healthcare), and γ-³²P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).