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. 2013 Mar 11;210(3):517–534. doi: 10.1084/jem.20120572

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Figure 4. — IL-27 down-regulates SPTBN1 during monocyte differentiation. (A) M-Mac, I-Mac, and macrophages treated with IFN-α (1 ng/ml) for 24 h (IFN-α Mac) were analyzed for gene expression levels of SPTBN1 by RT-PCR. (B) Whole-cell lysates of M-Mac, I-Mac, and IFN-α Mac were used for Western blotting. Protein expression levels of SPTBN1, SAMHD1, BST-2, and APOBEC3G were examined with specific antibodies. Samples were loaded in duplicate and β-actin served as an internal loading control. (C) M-Mac, I-Mac, and IFN-α Mac were infected with HIV-LUC-V. Data shown represent mean ± SE of three independent experiments. (D) M-Mac and I-Mac were analyzed for SPTBN1 expression on day 1, 3, 5, and 7 during differentiation. (top) Whole-cell lysates were subjected to Western blotting to examine protein levels of SPTBN1. (bottom) Gene expression levels of SPTBN1 were examined by RT-PCR. Relative mRNA levels of M-Mac and I-Mac were compared with that of undifferentiated monocyte on day 0, which is equal to 1. (E) Monocytes, M-Mac, and I-Mac derived from the same donor were infected with HIV-LUC-V. Values of luciferase activity were expressed relative to those obtained for M-Mac. Data shown represent mean ± SE of three independent experiments. (F) Monocytes, macrophages, dendritic cells, and CD4⁺ T cells obtained from the same donor were analyzed for SPTBN1 expression by Western blotting. MDDCs were induced by G-MCSF (50 ng/ml) and IL-4 (50 ng/ml) with or without IL-27 (50 ng/ml). CD4⁺ T cells were stimulated with PHA (5 µg/ml) and IL-2 (20 U/ml) with or without IL-27 (50 ng/ml).