Anti bcl 3 antibodies

Anti-Bcl-3 antibodies (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA)
were used as the primary antibodies. IHC analyses of diaminobenzidine
staining were performed using an HRP kit (UltraTek; Scytek, Logan, UT, USA).
The diaminobenzidine-stained specimens were visualized using a general
optical microscope with a camera (Carl Zeiss, Oberkochen, Germany).
Hematoxylin and eosin (H&E) staining of the tissue was conducted using a
MIRAX scan (Carl Zeiss). Images were processed with equivalent parameters
using ZEN Light Edition software (Carl Zeiss).

Treg cells from Bcl-3^TOE mice were differentiated, expanded and electroporated with Flag-tagged p50 constructs as in the section: Luciferase reporter Assays. Cells were re-stimulated with PMA and Ionomycin for 2 h and attached to cover glass slides with poly-L-lysine (SIGMA, P4832). Bcl-3 was stained with anti-Bcl-3 antibodies (Santa Cruz, sc-185 C-14) and p50 was visualized by staining for Flag tag with anti-Flag antibody (E. Kremmer, 6F7).

T_H0 and Treg cells were generated and expanded as described above and 1 × 10⁸ cells were lysed in 4 ml Meister Lysis Buffer (20 mM Tris/HCl pH 7.5, 0.25% NP40, 150 mM NaCl, 1,5 mM MgCl₂ and Protease Inhibitors (Roche, catalogue number: 04693132001) und 1 mM DTT). 60 μl Protein-G beads (Dynabeads Protein G, catalogue number: 10004D) were pre-coupled with 10 μg antibodies (anti-Bcl-3: Santa-Cruz, catalogue number: sc-185; anti-p50: Abcam, catalogue number: ab7971) in PBS and 0.05% Tween and then equilibrated in Meister Lysis Buffer and added to lysed cells and incubated for 4 h. Washing was performed with Lysis Buffer. Proteins were eluted with 80 μl 1 × SDS Lämmli loading dye, one-fourth was used for western blot analysis. Blottings were incubated with anti-p50 (Santa-Cruz, catalogue number: sc-8414) and anti-Bcl-3 antibodies (Santa-Cruz, catalogue number: sc-185).