Pd980509

For immunoblotting analysis, rabbit anti-phospho-p70S6K (S371), rabbit anti-phospho- p70S6K (T389), rabbit anti-GSK-3β, rabbit anti-phospho-GSK-3β (S9), rabbit anti-phospho-GS (S641), rabbit-anti-p38, rabbit-anti-phospho-p44/42 (T202/Y204), rabbit anti-phospho-p38 (T180/Y182) (Cell Signaling, Danvers, MA, USA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO, USA), and horseradish peroxidase-conjugate secondary anti-mouse and anti-rabbit Abs (Santa Cruz Biotechnology) were used. Rapamycin (0.2 µM, Sigma-Aldrich) or Torin 1 (0.5 µM Tocris, Bristol, UK) were added to DC culture 4 h after Mtb-infection for studying mammalian target of Rapamycin complex 1 (mTORC1) and mTOR inhibition respectively. For the selective inhibition of GSK-3β and p70S6K1 respectively, SB216763 (5 µM, Sigma-Aldrich) and PF4708671 (0.1 µM, Sigma-Aldrich) were used to treat DC 30 min before infection and Rapamycin treatment. To evaluate Rapamycin bystander effect, DC were treated for 30 min before infection and Rapamycin treatment with SB203580 (10 µM, Sigma-Aldrich) or SB202129 (10 µM, Sigma-Aldrich) to inhibit p38 or with PD980509 (0.1 µM, Sigma-Aldrich) to block p44/42.

Freshly isolated purified CD4⁺ T cells were rested overnight in RF1 and incubated in the presence or absence of either PI3K inhibitors LY294002 (50 µM) and Wortmannin (100 nM); p38 inhibitor SB203580 (5 µM); MEK1/ERK1/2 inhibitor PD980509 (50 µM); JNK inhibitor SP600125 (10 µM, All from Sigma-Aldrich); NF-κB inhibitors SC-514 (100 µM) and Bay 11-7082 (10 µM, Calbiochem, San Diego, CA); or AP-1 inhibitor SR11032 (1 µM, Tocris Biosciences, Bristol, UK) for 1 h. Cells (5 million) were treated with 100 nM of CCL19 for 15 min, lysed and immunoblotted as described above. Unstimulated or phorbol 12-myristate 13-acetate (PMA, 10 µg/ml Sigma-Aldrich) and ionomycin (2 µM, Sigma-Aldrich) stimulated cells were used as controls. Protein intensity was analysed using densitometry of gel images using FIJI software [47 (link)].