Rabbit anti p65 sc 372

ChIP assays were performed as described by Nelson et al. (2006) with minor modifications. In short, extracts from C666-1 cells were sonicated using an S-4000 sonicator (Misonix, Farmingdale, NY, USA) and incubated overnight with 5 µg of rabbit anti-p50 (sc-7178, Santa Cruz), 5 µg of rabbit anti-p65 (sc-372, Santa Cruz), or 5 µg of rabbit control IgG (ab46540, Abcam, Cambridge, Cambridgeshire, UK), and antibody–protein–DNA complexes were then pulled-down using Dynabeads Protein A (Invitrogen). The levels of immunoprecipitated DNA were determined by qPCR using a primer pair that amplified the Qp region, including the putative NF-κB site, and a control primer pair (Table 1).

Microscope coverglasses (Marienfeld GmbH & Co. KG, Lauda-Königshofen, Baden-Württemberg, Germany) with a diameter of 12 mm were coated with 10% poly-l-lysine (Santa Cruz) in water and washed three times with phosphate buffered saline (PBS) in a 24-well plate before cells were seeded onto them. The cells were fixed for 15 min in 4% paraformaldehyde in PBS. The fixated cells were washed two times with PBS and then immersed in 0.5% Triton X-100 in PBS for 3 min to permeabilize the cells, followed by three washes with PBS. The cells were blocked using PBS with 5% normal donkey serum (NDS) overnight at 4 °C, followed by incubation with rabbit anti-p65 (sc372, Santa Cruz) and mouse anti-β-tubulin (T8328, Sigma) at a 1:100 dilution in PBS with 5% NDS for 1 h at 37 °C. After three washes with 0.05% Tween-20 in PBS (PBST), the cells were incubated with secondary antibodies conjugated with different fluorophores at a 1:200 dilution in PBS with 5% NDS for 30 min at room temperature. Finally, the cells were washed three times with PBST, and then the cover glass was transferred onto a small drop of VECTASHIELD antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) on a microscope slide. The cells were examined with a confocal LSM (laser scanning microscope) 710 microscope (Carl Zeiss, Oberkochen, Baden Württemberg, Germany).