Macrophage Polarization Assay by Western Blot

To estimate the polarization of macrophages induced by PMA, lipopolysaccharide(LPS), interferon-γ(IFN-γ), IL-4, and IL-13, the expression of the markers of M1-type macrophages, CD86 and inducible nitric oxide synthase(iNOS), and the markers of M2-type macrophages, CD163 and IL-10, were detected by Western blotting. The influence of AFP on the expression of these marker proteins and PI3K/Akt signaling pathway-related proteins was analyzed in M0-type macrophages. M0 macrophages were infected with the AFP-expressed lentiviral vectors and treated with the PI3K inhibitor Ly294002 (final concentration:20 μM) for 48 h. The expression of these proteins was analyzed by Western blotting. Briefly, these proteins were probed with the following primary antibodies: mouse anti-CD86 (1:500), anti-iNOS (1:500), anti-CD163 (1:500), anti-IL-10 (1:500), anti-β-actin (1:1000), rabbit anti-PI3K (1:400), anti-Akt (1:400), or anti-p-Akt(Ser473) (1:400) (all from eBioscience and Abcam Inc.). The detailed procedure has been previously described (20 (link), 28 (link)).