TGFβ regulation of TRAF1 levels in T cells. (a) Mice were infected with LCMV clone 13 for 21 d. 200 µg/mouse of anti-TGFβ1 or control antibody was injected i.p. on day 21. On day 24, CD8 T cells were purified from total splenocytes, stained, and sorted for tetramer⁺PD-1⁺ CD8 T cells (GP33 + GP276 + NP396) and subjected to Western blot analysis. Left panel shows a representative blot, and the right panel shows the summary of results with each symbol representing a single mouse from the same experiment. A similar increase in TRAF1 upon TGFβ blockade was obtained in two additional smaller experiments. (b) Splenocytes from OT-1 mice were stimulated with SIINFEKL peptide for 36 h with or without TGFβ. Live CD8 T cells were purified and lysed for Western blotting for TRAF1, TRAF2, and Actin. Data are representative of 3 experiments. (c) Splenocytes from OT-1 mice were stimulated with SIINFEKL peptide for 20 h with or without TGFβ, followed by addition of 1 µg/ml of CHX in the continued presence of TGFβ. Note that if TGFβ was added at the same time as CHX, without the pretreatment, it did not cause loss of TRAF1 (not depicted). Cells were harvested at 0, 1, 2, 3, and 4 h after drug treatment. Live CD8 T cells were purified, and then subjected to Western blot. Data are representative of two experiments. (d) Same as in c, except that 25 nM of chloroquine (Clq) or 25 nM of lactacystin (Lac) was added to the cells at the time of CHX addition, where indicated. Cells were harvested and purified at 2 h after drug treatment. Data are representative of two experiments.