FIGURE 3.

L-kynurenine promotes the activation of c-Src via AhR. (A) Immunoprecipitation of AhR from SYF cells, either reconstituted with c-Src or empty vector, and detection of Src and AhR by sequential immunoblotting with specific antibodies. Cells were treated with L-kynurenine for 30 and 60 min before lysis. Input refers to whole-cell lysates used as control of protein expression. One representative immunoblot of three is shown. (B) Expression of pSrc, Src, and AhR in nuclear and cytoplasmatic extracts from SYF cells expressing Src and treated as in (A). Lamin-β and β-actin were used as loading control of nuclear and cytoplasmic extracts, respectively. One representative experiment of three is shown. (C) Immunoprecipitation of AhR from cDCs either unprimed or primed with LPS and then exposed to L-kynurenine for 30 and 60 min. Src and AhR protein expression were analyzed by sequential immunoblotting with specific antibodies. Input refers to whole-cell lysates used as control of protein expression. One representative experiment of three is shown. (D) Expression of pSrc, Src, and AhR in nuclear and cytoplasmatic extracts from cDCs treated as in (C) for 60 min. Lamin-β and β-actin were used as loading control of nuclear and cytoplasmic extracts, respectively. One representative experiment of three is shown. Src/AhR ratio (A,C) is calculated by densitometric quantification of the specific bands and is reported as fold change against untreated cells. pSrc/Src ratio [(B,D); left panel, cytosol] and Ahr/Lamin-β ratio [(B,D); right panel, nucleus] are measured by densitometric quantification of the specific bands and are expressed relative to untreated cells. Ratios are means from the three experiments. *P < 0.05 (one-way ANOVA).