Anti p100 p52

Birinapant, CompA, necrostatin, and IDN-6556⁵⁰ were gifts from TetraLogic Pharmaceuticals. Fc-TNF was generated in-house. Q-VD-OPh (Q-VD) was purchased from MP Biomedicals. Docetaxel was purchased from ActiveBiochem. Antibodies used for neutralization assays anti-TNF (MAB610) and anti-TRAIL (MAB375) were purchased from R&D Systems. Antibodies used for immunoblotting and immunohistochemistry were purchased as follows: anti-cIAP1 (1E1-1-10) and anti-cIAP2 (16E-6-3) from Enzo, anti-XIAP (2F1) from MBL, anti-cleaved caspase-3 (Asp175) from Cell Signaling Technology), anti-NIK (Cell Signaling Technology), anti-p100/p52 (Cell Signaling Technology), anti-tubulin (DM1A) and anti-actin (AC-15) from Sigma-Aldrich, and anti-KI67 (Thermo Fisher Scientific).

Recipient splenic lymphocytes were obtained using lymphocyte separation solution (Dakewe Biotech Co., Ltd.) according to the manufacturer’s instructions. The lymphocytes were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor. Lysates were centrifuged at 12,000 g and 4°C for 25 min, and the supernatant was subsequently collected. Total proteins were quantified by bicinchoninic acid assay. Thereafter, 20 µg proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. After being blocked with 5% milk for 1 h, the membrane was incubated at 4°C overnight with the following primary antibodies (all from Cell Signaling Technology, Inc.): anti-P100/P52 (1:1,000; #4882), anti-RelB (1:1,000; #10544), and anti-β-actin (1:1,000; #3700). The membranes were washed using Tris-buffered saline with Tween-20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; Abcam) at room temperature for 1 h. The proteins were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). Band intensity was analyzed using ImageJ software and normalized to the value of β-actin.

Malt1^+/− and Malt1^−/− MEFs were obtained from Jürgen Ruland (Technische Universität Munich, Munich, Germany). Malt1^+/− and Malt1^−/− MEFs were stimulated with 100 ng/ml murine soluble TNF (mTNF) (R&D Systems). Cytoplasmic and nuclear extracts were prepared using a nuclear protein extraction kit according to the manufacturer's instructions (Active Motif, Belgium). Immunoblots were probed with primary anti-p65 (Santa Cruz Biotechnology), anti-RelB (Cell Signaling), and anti-p100/p52 (Cell Signaling).