Western blot was performed using Anti-MIF Polyclonal Antibody (Product # PA5-81446) and a 15 kDa band corresponding to Macrophage migration inhibitory factor was observed across cell lines and tissues tested. The MIF protein expression was upregulated in U-87 MG upon VEGF treatment. Whole cell extracts (30 µg lysate) of Jurkat (Lane 1), HUVEC (Lane 2), RAW 264.7 (Lane 3), HEK-293 (Lane 4), Mouse Brain (Lane 5), Rat Spleen (Lane 6) in Fig. a; THP-1 (Lane 1), U-937 (Lane 2), U-87 MG (Lane 3), U-87 MG treated with VEGF (100 ng/mL for 48hrs) (Lane 4) in Fig. b, were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) 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 (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:6000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).