Ab31412

TCam-2 cells were harvested after 48 h of transfection for cytospin preparation using Shandon Cytospin 3 centrifuge (Thermo Scientific, Waltham, MA, USA). The cytospun cells were washed with cold PBS twice before fixation with 4% paraformaldehyde (USB Corporation/Affymetrix, Santa Clara, CA, USA) for 10 min at room temperature. Cells were permeabilized with cold solution of 0.5% Triton-X for 10 min followed by overnight incubation at 4 °C with anti-PEG3 (ab139166; Abcam; 1:200), anti-p105/p50 (ab31412; Abcam; 1:50) or anti-p100/p52 (3017S; Cell Signaling Technology; 1:100) primary antibodies. Alexa Fluor 488 and 546-conjugated secondary antibodies (A11008 and A11003, respectively; Life Technologies) were diluted to 1:400 and applied on the cells for an hour at room temperature. Cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) in Vectorshield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured in a confocal microscope Leica TCS SP5 (Lecia Microsystems GmbH, Wetzlar, Germany).

Immunohistochemistry was performed according to previously described methodology ^{44 (link)}, using rabbit polyclonal anti-PEG3 (HPA026070; Sigma-Aldrich) and anti-NF-κB p105/p50 (ab31412; Abcam) antibodies at dilutions 1 :50 and 1:100, respectively. Briefly, TMA slides were de-waxed with xylene and re-hydrated in a descending ethanol series before quenching with 3% hydrogen peroxide (Merck, Darmstadt, Germany) for 20 min. The antigen retrieval was performed with citrate buffer pH 6.0 (S2369; Dako, Glostrup, Denmark) at 95–99 °C for 20 min. Slides were incubated with primary antibodies at 4 °C overnight. The REAL EnVision Detection System Peroxidase/DAB+ (K5007; Dako) was used and slides were counterstained with Mayer’s hematoxylin (Histolab Products AB, Gothenburg, Sweden). The immunostaining was classified as negative or positive (weak, intermediate and strong) based on the scoring of the nuclear staining intensity (0, 1+, 2+ or 3+).