c-Src kinase is involved in the tyrosine phosphorylation and activity of SLC11A1 in differentiating macrophages
Fig 7
Identification of the tyrosine phosphorylation site of SLC11A1 by c-Src.
(A) Site-directed mutagenesis was used to make the Tyr to Phe substitution at position 15 or 38 of SLC11A1. (B) U937 cells stably expressing c-Myc-tagged wild-type or mutated SLC11A1 (Y15F or Y38F) were transiently transfected with a pCB6-Src vector. 24 hours after transfection, cells were treated with PMA for 48 hrs. Cell lysates were prepared and immunoprecipitated with anti-c-Myc antibody (9E10). The tyrosine phosphorylation of SLC11A1was analyzed by immunoblotting with antibody 4G10 to phosphotyrosine (top panel). The Western blots were stripped and re-probed with an antibody against SLC11A1 (middle panel). The expression of c-Src (bottom panel) was also analyzed using anti-Src antibody. (C) The level of SLC11A1 phosphorylation in transfected cells was quantified by densitometry analysis and normalized to the level of total SLC11A1 protein. The relative phosphorylation level of the WT SLC11A1 in U937 cells was set as 100%. Data represented as mean ± S.E.(n = 3). ***P <0.001, compared with WT SLC11A1.