Rapa increases expression of p27^KIP1 protein.

(A-B) DCs were cultured with IL-4 and GM-CSF in the absence or presence of Rapa (10⁻⁷ M) and either with or without addition of FK506 (5 × 10⁻⁶ M) for 48 hours. Part of the cells was used to prepare whole cell lysates. Equal amounts of protein (20 μg/lane) were loaded, and the levels of p27^KIP1 were determined (A). Apoptosis was detected in remaining cells by flow cytometry, using annexin V–FITC and PI staining (B). Data shown are representative for 3 independent experiments performed with different donors. (C-D) DCs were cultured in IL-4 and GM-CSF with or without addition of 1 μM Rapa. Cells were harvested after 5 hours, 24 hours, or 48 hours of incubation. Whole cell lysates (20 μg /lane) were loaded, and the levels of p27^KIP1 were determined by Western blot analysis (C). The relative expression of p27^KIP1 was determined by using Stratagene-EagleSight (La Jolla, CA) and represents the ratio of each band from Rapa-treated DCs to that of untreated DCs. Results are expressed as mean ratio (± SD) of 4 independent experiments (D).