Fig. 6

MIF interacts with Aβ oligomers. a 0.2 nmol of Aβ oligomers and 0.1 nmol of purified GFP protein were spot on a nitrocellulose membrane, and the membrane was incubation with mixed proteins of recombinant hMIF and purified GFP at the concentration of approximated 5 μM at 4 °C for overnight. The membrane was then subjected to immunoblotting to detect MIF and GFP by a monoclonal anti-MIF antibody and a polyclonal anti-GFP antibody, respectively. The red channel detects IR-dye-labeled goat anti-rabbit antibody, and the green channel detects IR-dye-labeled goat anti-mouse antibody. b Brains from 4-month-old APP23/PS45 and wildtype (as controls) mice were homogenized in 5x PBS with 0.5% Triton-100 (v/w) and centrifuge at 16,000×g at 4 °C for 15 min. The supernatants were collected and subjected to ultracentrifugation at 100,000×g for 1 h at 4 °C, and the second supernatants were collected and labeled as S′. Homogenates from each layer were further dissolved in RIPA-DOC buffer followed by brief sonication. The same amount of protein was loaded on 16% and 12% Tris-tricine SDS PAGE gels for Aβ and MIF separation, respectively. Aβ was detected by a 6E10 antibody, MIF was detected by anti-MIF antibody, CTFs were detected by a C20 antibody to confirm the expression of the transgene, and β-actin was detected by β-actin antibody serving as the loading control. S′, supernatant after ultracentrifugation; P1, pellet 1; P2 pellet 2. c Quantification of the level of MIF protein from b. Values represent mean ± SEM, n = 3. *P < 0.05 by Student’s t test