4.8. Western blot

To assess protein levels of KLF-4, PPAR-γ and ORM-1 in whole-cell lysates of human monocyte-derived Mφ-differentiated +/−sCD83, Western blot analyses were performed. Whole-protein lysates (30 µg per lane) were separated via SDS polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane (GE Healthcare). After blocking in blocking reagent (5% BSA-TBST), membranes were incubated with the following primary antibodies overnight (4°C): mouse-anti-human KLF4 (Cat. No.: sc-166100; Clone: B-9, Santa Cruz), mouse-anti-human PPAR-γ (Cat. No.: MA5-15417; Clone: 3A4A9, 1E6A1; Invitrogen), and mouse-anti-human ORM-1 (Cat. No. MA5-41544; Clone: D1; mouse β-actin (Clone: AC-74, Sigma-Aldrich). Specific signals were detected using the appropriate HRP-labeled secondary antibody and the ECL Prime Western Blotting Detection Reagent (GE Healthcare). Quantification of Western blots was performed using the ImageJ/Fiji software (53). The intensities of bands are visualized in bar graphs and represent the protein amount in arbitrary units. Band intensities of KLF-4, PPARγ, and ORM-1 were normalized to β-actin that served as a loading control.