Immunofluorescence analysis of Macrophage migration inhibitory factor was performed using 70% confluent log phase U-87 MG cells treated with VEGF (100 ng/mL for 48hrs). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with MIF Polyclonal Antibody (Product # PA5-95222) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic and secreted localization. Panel e represents untreated U-87 MG cells with lower expression level of MIF. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Western blot was performed using Anti-MIF Polyclonal Antibody (Product # PA5-95222) and a 15 kDa band corresponding to Macrophage migration inhibitory factor was observed across cell lines tested and was upregulated in U-87 MG upon VEGF treatment. Whole cell extracts (30 µg lysate) of 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), HEK 293 (Lane 5) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). 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 (0.5 µg/mL) 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).