b'AntibodiesAntibodies for studying the role of MIF in inflammatory response and angiogenesisMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokineApart from its canonical role as an immunoregulator, MIF also plays a involved in inflammatory and immune responses. Although it wasrole in cell proliferation and differentiation, and its aberrant expression initially identified as a T cellderived lymphokine that inhibited thehas been seen in many cancer tissues. MIF has also been shown random migration of macrophages, further studies have shown MIFto play a role in neovascularization, based on studies of lymphoma, to be ubiquitously expressed across different organs and tissues.melanoma, and colon cancer cells. A strong correlation of MIF MIF expresses in response to the immunosuppressive effects ofexpression exists with vascular endothelial growth factor (VEGF), the glucocorticoids and counter-regulates the inhibition of inflammatoryprimary angiogenic factor in glioblastomas, suggesting the two may cytokines including TNF-, IL-1, and IL-6 in macrophages andshare co-regulatory pathways (Figure 4).T lymphocytes. MIF secretion by macrophages is induced by endotoxins such as lipopolysaccharide (LPS) and is further elevated by cytokines including TNF- and IFN-. Serum levels of MIF remain elevated 220 hours after LPS administration, which highlights the roleBof MIF in host inflammatory response.U-87 MGA THP-1 U-937 Jurkat HEK293260160110806050403020MIF15 ~15 kDa10GAPDHRecombinant VEGF,+ 100 ng/mL for 48 hrFigure 4. Specificity of the MIF antibody observed when MIF is upregulated in response to VEGF treatment in the glioblastoma cell line U-87 MG. (A) Western blot (WB) analysis was performed using the Invitrogen MIF Monoclonal Antibody (2Ar3) (Cat. No. MA1-20881), and a 15 kDa band corresponding to MIF was observed across cell lines tested. Upregulation of MIF expression was observed in U-87 MG cells after VEGF treatment (100 ng/mL for 48 hr). Whole cell extracts (30 g lysate in all lanes) of the indicated cell lines were electrophoresed and transferred onto a nitrocellulose membrane, which was probed with the primary antibody (1 g/mL) and detected by chemiluminescence with the Invitrogen Goat AntiMouse IgG (H+L), Superclonal Recombinant Secondary Antibody, HRP conjugate (Cat. No. A28177, 1:6,000 dilution), using the Invitrogen iBright FL1500 Imaging System (Cat. No. A44115). Chemiluminescent detection was performed using the Thermo Scientific Pierce ECL Western Blotting Substrate (Cat. No. 32106). (B) Immunofluorescence (IF) analysis using the Invitrogen MIF Polyclonal Antibody (Cat. No. PA5-81446, 1:100 dilution) shows upregulation of MIF expression in U-87 MG cells upon VEGF treatment. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Invitrogen Triton X-100 Solution (Cat. No. HFH10) for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with the MIF Polyclonal Antibody (Cat. No. PA5-81446) at a 1:100 dilution in 0.1% BSA, incubated at 4C overnight, and then labeled with the Invitrogen Donkey AntiRabbit IgG (H+L), Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Cat. No. A32790, 1:2,000 dilution), for 45 minutes at room temperature. Panel A: VEGF treated (green). Panel B: Nuclei stained with Invitrogen ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962, blue). Panel C: F-actin stained with Invitrogen Rhodamine Phalloidin (Cat. No. R415, 1:300, red). Panel D: Merged image showing cytoplasmic and secreted localization. Panel E: Untreated U-87 MG cells with lower expression level of MIF. Panel F: Control cells with no primary antibody to assess background. The images were captured at 60x magnification.Search our wide selection of antibodies at thermofisher.com/antibodiesContents Inside the Cell//Issue No. 12 17'