P38 mapk inhibitor sb203580

Epidermal cells isolated from day 1 neonatal mice were stimulated on 24-well plates (1 × 10⁶ cells/well) coated with 10 μg/mL anti-TCR Cδ mAb in IMDM supplemented with 10% FCS, 50 μM 2-mercaptoethanol, and 10 ng/mL recombinant mouse IL-2 for 5 days at 37°C. Various inhibitors of signaling pathways downstream of TCR were added during this period. Cells were harvested and rested on uncoated plates in the same culture medium without inhibitors for 2 days at 37°C to allow the recovery of TCR expression before restimulation with PMA/ionomycin followed by intracellular cytokine staining.
Optimal concentrations of the inhibitors were predetermined as the maximum concentrations that did not affect viable Vγ3⁺ T-cell yields after the cultures. The following inhibitors were used at the indicated concentrations: calcineurin inhibitor cyclosporin A (0.01 μM; Cell Signaling Technology), MEK1/2-ERK1/2 inhibitor U0126 (5 μM, Cell Signaling Technology), p38 MAPK inhibitor SB203580 (10 μM, Cell Signaling Technology), JNK inhibitor SP600125 (5 μM, Cell Signaling Technology), PKCθ inhibitor sotrastaurin (0.1 μM; Abcam, Cambridge, UK), PI3K inhibitor LY294002 (5 μM, Cell Signaling Technology), mTORC1 inhibitor rapamycin (5 ng/mL, Sigma–Aldrich), and mTORC1/2 inhibitor Torin 1 (0.05 μM, Cell Signaling Technology).

We investigated whether the MAPK and NF-κB signal pathways were involved in Ang II–induced cytokine expression. ARPE-19 cells were seeded in 24-well plates to become confluent. After being serum starved for 24 hours, cells were pre-incubated with p38MAPK inhibitor SB203580 (Cell Signaling Technology, Danvers, MA), JNK inhibitor SP600125 (Cell Signaling Technology, Danvers, MA), ERK1/2 inhibitor PD98059 (Cell Signaling Technology, Danvers, MA) and NF-κB inhibitor BAY11-7082 (Sigma-Aldrich, St. Louis, MO) at the concentration of 10 μM for 30 minutes. Subsequently, Ang II (10⁻⁶ M) was added to the medium. After 48 hours of incubation, the supernatants were collected and stored at −80 °C until analysis. Total RNA of the cells after treatment was extracted for further assay.