FIG. 3.

Role of JAK2/STAT signaling in SIRPα-induced NO production in NR8383 macrophages. (A) Tyrosine phosphorylation in macrophages stimulated for 5 min with control IgG1 (MAb BF5, 20 μg/ml) or anti-SIRPα MAb ED9 (20 μg/ml). Total lysates of NR8383 cells subjected to Western blotting and staining with anti-PY MAb 4G10. Note that SIRPα ligation promotes tyrosine phosphorylation of several cellular proteins (arrows). (B) Immunoprecipitates (IP) of SIRPα in NR8383 cells after stimulation by anti-SIRPα MAb ED9 (10 μg/ml) or total lysates (TL) were Western blotted (WB) and probed with anti-JAK2. Note that JAK2 associates with SIRPα in particular after SIRPα ligation by anti-SIRPα MAb ED9 (10 μg/ml). (C) Anti-SIRPα MAb ED9 (10 μg/ml)- or IFN-γ (20 U/ml)-stimulated NR8383 cells were lysed directly in SDS sample buffer. The panels show the same Western blot probed sequentially with anti-STAT1-PY, total anti-STAT1, and anti-iNOS antibodies. (D) Anti-SIRPα (MAb ED9, 20 μg/ml, 18 h of incubation)-induced NO production with or without 30 min of preincubation with AG490 (10 μM). Results from representative experiment that was performed in triplicate are shown (results are expressed as means ± SD).