The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection
Fig 2
ADAP enhances TGF-β1/TβRI signaling via TRAF6-TAK1-SMAD3.
(A) Wild type controls, ADAP-overexpressing Jurkat cells or ADAP deficient Jurkat cells (JDAP) were transfected with the (CAGA)12-luciferase and (SBE)4-luciferase reporters respectively. After exogenous TGF-β1 treatment (5ng/mL), the luciferase activity was assessed. Data are representative of two independent experiments. (B) Wild type or ADAP-/- CD8+ OT1 CTLs were generated with 10nM OVA257-264 peptide stimulation, then treated with 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of pSMAD3 (ser 423/425), total SMAD3 and β-actin were examined. The relative ratio between p-SMAD3 and β-actin was shown. Data are representative of two independent experiments. (C) WT and ADAP KO CD8+ CTLs were treated with 5ng/mL TGF-β1 for 30min, followed by preparation of cytoplasmic and nuclear extracts. Equal amounts of cytoplasmic and nuclear extracts were loaded for western blotting assay with antibodies against SMAD3. SP1 expression in nuclear extracts and tubulin expression in cytoplasmic extracts were used as controls. Data are representative of two independent experiments. (D) Wild type or ADAP-/- CD8+ OT1 Tg cells were stimulated with 10nM OVA257-264 peptide and 5ng/mL exogenous TGF-β1 as the indicated time points. The levels of p-p38, p-JNK, p-p85 (Tyr458), p-AKT (Ser473) and β-actin were examined. Data are representative of two independent experiments. (E) Cell lysates from OT-I CD8+ CTLs were immunoprecipitated with anti-TRAF6 antibody, followed by immunoblotting with antibodies against ADAP, TAK1 and TRAF6. Data are representative of two independent experiments. (F)The mRNA levels of TGF-β1 were examined in CD8+ CTLs from wild type or ADAP-/- OT1 Tg mice that were generated with 10nM OVA peptide and 5ng/mL exogenous TGF-β1 in the presence or absence of the 2uM TAK1 inhibitor 5Z-7-oxozeaenol. Data are representative of three independent experiments.