Fig 1: Pref-1 inhibits MIF secretion by interaction with the cell membrane protein, integrin 1ß, and inhibiting p115, a cofactor for MIF release(A) 3T3-L1 undifferentiated (3T3U) Pref-1+ cells were treated with or without rPref-1 (2.5 µg/ml) in the absence or presence of LPS (5 µg/ml) for 24 h p115 was then quantified in cytosol and medium by western blot.(B) MIF (green) and p115 (red) were visualized by immunofluorescence staining in 3T3-L1 undifferentiated Pref-1+ cells following vehicle or LPS treatment. The nucleus was stained by DAPI (blue), (Scale bars, 10 µm).(C) MIF release from 3T3-L1 undifferentiated Pref-1+ cells following vehicle or LPS treatment in the presence or absence of rPref-1.(D) p115 in cytosol and medium of 3T3-L1 differentiated adipocytes (3T3D) quantified by western blot following rPref-1 treatment for 24 h.(E) p115 release from WATs isolated from NC and PD groups in the presence of absence of rPref-1 (2.5 µg/ml).(F) The gene expression of ITGB1 and ITGA5 in Pref-1+ cells and adipocytes isolated from WT visceral adipose tissue.(G) ITGB1 and ITGA5 genes in 3T3U and 3T3D.(H) In 3T3U and 3T3D, immunoprecipitation was performed with negative-control antibody (IgG) or anti-ITGB1 following the treatment of rPref-1. Immunoprecipitated proteins were then detected by anti-Pref-1 antibody.(I–K) MIF and p115 release from 3T3U and 3T3D following the treatment of rPref-1, in the presence or absence of integrin inhibitor, RGDS peptide (100 µM).(L) Following normal chow (NC) or high palmitic acid diet (PD) feeding, visceral adipose tissues isolated from WT mice were initially immunoprecipitated with anti-ITGB1 antibody and the immunoprecipitated proteins were further detected by anti-Pref-1 antibody.All data are presented as mean ± SD. *p = 0.05 increase and #p = 0.05 reduction vs. Vehicle, Pref-1+ cells, IgG, NC, or all other groups. The n.s. represents no significance.