FIGURE 7.

IFNAR1 palmitoylation does not affect Tyk2 association to IFNAR1 or Jak kinases activation. A, Tyk2 is constitutively associated with IFNAR1 independently of its palmitoylation status. BRET saturation curves were obtained by measuring BRET in CHO cells expressing fixed quantities of BRET donor (Rluc-Tyk2) and increasing amounts of BRET acceptors (carboxyl-terminally YFP-tagged IFNAR1 CC, IFNAR1 AC, or IFNGR1). Relative amounts of BRET acceptor are expressed as the ratio between the fluorescence of the acceptor over the luciferase activity of the donor. YFP° corresponds to background fluorescence in cells expressing the BRET donor alone. BRET ratio values were from 26 individual transfections and were grouped as a function of the amount of BRET acceptor (mean ± S.D.; n = 4). B, Jak kinases are still fully activated even in absence of IFNAR1 palmitoylation. L929R2 cells stably transfected with wild-type IFNAR1 or the AC mutant were stimulated or not with 1000 units/ml IFN-α for the indicated times. Cell lysates were analyzed by Western blotting against tyrosine-phosphorylated Tyk2 (pTyk2), tyrosine-phosphorylated Jak1 (pJak1), and Jak1 as indicated. The figure shown is representative of three independent experiments. Quantification was made using ImageJ software, and mean ± S.D. of at least three independent experiments are presented. Results correspond to the ratio between the amount of pTyk2 or pJak1 and Jak1 amount normalized to the control without IFN. Significant differences were evaluated by using a Student's t test.