Adezmapimod (SB203580)

4 μg of sheep anti-PKBα is immobilized on 25 μL of protein G-Sepharose overnight (or 1.5 hours) and washed in Buffer A (50 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 0.5 mm Na₃VO₄, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, pepstatin, leupeptin, and 1 μm microcystin). The immobilized anti-PKB is then incubated with 0.5 ml of lysate (from 5 × 10⁶ cells) for 1.5 hours and washed three times in 0.5 mL of Buffer A supplemented with 0.5 m NaCl, two times in 0.5 mL of Buffer B (50 mm Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mm EGTA, and 0.1% β-mercaptoethanol), and twice with 100 μl of assay dilution buffer; 5× assay dilution buffer is 100 mm MOPS, pH 7.2, 125 mm β-glycerophosphate, 25 mm EGTA, 5 mm sodium orthovanadate, 5 mm DTT. To the PKB enzyme immune complex is added 10 μL of assay dilution buffer, 40 μm protein kinase A inhibitor peptide, 100 μm PKB-specific substrate peptide, and 10 μCi of [γ-³²P]ATP, all made up in assay dilution buffer. The reaction is incubated for 20 minutes at room temperature with shaking, then samples are pulse spun, and 40 μL of reaction volume are removed into another tube to which is added 20 μL of 40% trichloroacetic acid to stop the reaction. This is mixed and incubated for 5 minutes at room temperature, and 40 μL is transferred onto P81 phosphocellulose paper and allowed to bind for 30 seconds. The P81 piece is washed three times in 0.75% phosphoric acid then in acetone at room temperature. γ-³²P incorporation is then measured by scintillation counting.

CT6 cell and BA/F3 F7 cell are rested by washing three times in RPMI and culturing overnight in RPMI, 5% fetal calf serum in the absence of growth factor, antibiotics, or β-mercaptoethanol supplements. 2–5 × 10⁶ rested CT6 cells are resuspended in 2 mL of RPMI, 5% fetal calf serum and preincubated with SB203580 or vehicle control as indicated in figure legends. Cells are then stimulated with 20 ng/ml recombinant human IL-2 for 5 minutes at 37  °C and pelleted in a minifuge for 30 seconds, medium is aspirated, and the pellet is lysed in the appropriate buffer. BA/F3 cells stably expressing deletion mutants of IL-2 receptor β chain are maintained in glutamine containing RPMI further supplemented with 5% fetal calf serum and 0.2 μg/mL G418. The cells are then washed extensively, rested overnight, and washed again before activating with IL-2; such cell preparations are >90% T cells. Cellular proliferation assays are performed by measurement of [³H]thymidine incorporation.