Western blot was performed using Anti-TNFAIP3 Polyclonal Antibody (Product # PA5-17149) and a 89 kDa band corresponding to TNFAIP3 was observed across all cell lines and tissue extracts tested and was upregulated upon IL-1 alpha, TNF-alpha and LPS treatments. A uncharacterized band (*) was also observed in some cell lines at ~32 kDa. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with IL-1 alpha (20 ng/mL for 4H) (Lane 2), HeLa treated with TNF-alpha (20 ng/mL for 4H) (Lane 3), THP-1 (Lane 4), RAW 264.7 (Lane 5), RAW 264.7 treated with LPS (1 µg/mL for 6H) (Lane 6), Jurkat (Lane 7) and Mouse Spleen (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1,000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).