Western blot was performed using Anti-IRAK4 Polyclonal Antibody (Product # PA5-17531) and a 52 kDa band corresponding to IRAK4 was observed across cell lines tested. Whole Cell Extract (40 µg lysate) of THP-1 (Lane 1), THP-1 treated with PMA (25 nM for 48 hours) and LPS (100 ng/ml for 24 hours) (Lane 2), THP-1 (Lane 3), THP-1 subjected to M0 macrophage differentiation and treated with LPS (100ng/ml for 12 hours) (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). THP-1 differentiated macrophage upon LPS treatment are reported to show down regulation in IRAK4 levels (doi:10.1016/j.cellsig.2008.10.009).