Ab10297

Co-immunoprecipitation (Co-IP) was performed as previously described [9 (link), 12 (link), 14 (link)], with antibodies specific for hnRNPU (ab10297), CTCF (ab188408), FLAG (ab125243), or Myc (ab9106, Abcam Inc.). Bead-bound proteins were released and analyzed by Western blot.

Cell samples were washed with precooled 0.1 M PBS and then lysed with cell lysis solution (RIPA). Ice bathing was conducted for 30 min, followed by centrifugation at 12000 rpm at 4°C for 10 min. The supernatant was collected and preserved at –20°C for further use. BSA (2 μg/μl) was diluted with 0.1 M PBS to the following concentrations: 20, 15, 10, 5, 2.5, and 0 μg/ml. Protein concentration was detected using BCA (Thermo Fisher Scientific Inc., MA, U.S.A.) according to the instructions and sample numbers. The protein was electrophoresed in a 4°C chromatography cabinet with 80 V of compressive gel and 120 V of separation gel. The electrophoresed proteins were transferred on to a PVDF membrane, blocked in TBST (25 mM Tris, 140 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% skimmed milk and incubated for 2 h. The proteins were combined with the primary antibody of p120 (1:500; article number ab10297; ABCAm Inc., Cambridge, MA, U.S.A.) and β-actin (1:10000; article number ab8226; ABCAm Inc., Cambridge, MA, U.S.A.) and incubated at 4°C overnight. After being washed (3 × 10 min) with TBST, the secondary antibody was added and incubated at room temperature for 1 h. Subsequently, the cells were rewashed with TBST (3 × 10 min) and chemiluminescence was conducted. The results were analyzed according to the developed and fixed X-ray films.