Figure 3.
Figure 3. Nef-induced FasL induction requires p38 MAPK activation. (A) Nef induced phosphorylation of p38. Western blot analysis of protein extracted from Jurkat cells transfected with 5 μg vector control or pNef in the presence or absence of p38 inhibitor-II. Samples were prepared as described in “Materials and methods” and resolved on 12% SDS-PAGE gels. Gels were transferred to PVDF membranes and immunoblotted with p38MAPK- and phospho-p38MAPK-specific antibodies as indicated. Nef induced phosphorylation of p38, and this activation can be blocked by the p38 inhibitors (p38 inhi). (B-C) Inhibition of Nef-induced FasL promoter activity by a dominant-negative p38. Jurkat cells were transfected with plasmid containing pVector, the hFasL-Luc promoter, the pNef plus hFasL-Luc promoter, or the pNef plus hFasL-Luc promoter together with wild-type p38 (B) or p38-DN (C) plasmid constructs as indicated. Luciferase activity in whole-cell lysates was assayed after 12 to 18 hours and is shown as the mean value ± SEM. Transfection efficiency was monitored by cotransfection of a plasmid encoding β-gal and results were normalized to β-gal levels. Similar results were obtained in 3 independent experiments and were reproducible. Nef strongly induced the FasL promoter activation (B). In contrast, induction of FasL transcriptions was reduced in cells cotransfected with dominant-negative mutant p38 (C). (D) Construction and expression of p38siRNAs. Four different target siRNA expression vectors were constructed as described in “Materials and methods.” Jurkat T cells were transiently transfected with 5 μg siRNA control vector or p38siRNA expression constructs as indicated clones. Cell lysates were extracted 48 hours after transfection, and immunoblotting was performed by using an anti-p38 antibody and an actin antibody as a control for equal loading. p38 expression was suppressed significantly in cells transfected with clone p38-61 and moderately in cells transfected with the other clones compared with control vector transfected. (E) FasL expression is significantly reduced by p38siRNA. Jurkat T cells were electroporated with 5 μg plasmid-expressing vector, pNef, or p38siRNA (clone p38-61) and pNef plasmids as indicated. At 48 hours after transfection, the surface levels of FasL expression were determined by flow cytometry using a FasL-specific antibody. Filled histograms show the FasL expression, and open histograms represent isotype-matched control antibodies. p38 siRNA inhibited Nef-induced FasL induction. Similar results were obtained in 3 independent experiments. Transfection efficiency was monitored by cotransfection of a pCMV plasmid encoding GFP, which also served as a marker for gating on transfected cells. (F) p38 siRNA does not affect Nef expression. Total protein extracts were prepared from the groups transfected with vector, pNef, or p38siRNA plus pNef. Of each protein sample, 50 μg was separated on 12% SDS-PAGE and analyzed by Western blot with a polyclonal antibody against Nef antibody. Immunoblotting was also performed using an antiactin antibody as an internal control.

Nef-induced FasL induction requires p38 MAPK activation. (A) Nef induced phosphorylation of p38. Western blot analysis of protein extracted from Jurkat cells transfected with 5 μg vector control or pNef in the presence or absence of p38 inhibitor-II. Samples were prepared as described in “Materials and methods” and resolved on 12% SDS-PAGE gels. Gels were transferred to PVDF membranes and immunoblotted with p38MAPK- and phospho-p38MAPK-specific antibodies as indicated. Nef induced phosphorylation of p38, and this activation can be blocked by the p38 inhibitors (p38 inhi). (B-C) Inhibition of Nef-induced FasL promoter activity by a dominant-negative p38. Jurkat cells were transfected with plasmid containing pVector, the hFasL-Luc promoter, the pNef plus hFasL-Luc promoter, or the pNef plus hFasL-Luc promoter together with wild-type p38 (B) or p38-DN (C) plasmid constructs as indicated. Luciferase activity in whole-cell lysates was assayed after 12 to 18 hours and is shown as the mean value ± SEM. Transfection efficiency was monitored by cotransfection of a plasmid encoding β-gal and results were normalized to β-gal levels. Similar results were obtained in 3 independent experiments and were reproducible. Nef strongly induced the FasL promoter activation (B). In contrast, induction of FasL transcriptions was reduced in cells cotransfected with dominant-negative mutant p38 (C). (D) Construction and expression of p38siRNAs. Four different target siRNA expression vectors were constructed as described in “Materials and methods.” Jurkat T cells were transiently transfected with 5 μg siRNA control vector or p38siRNA expression constructs as indicated clones. Cell lysates were extracted 48 hours after transfection, and immunoblotting was performed by using an anti-p38 antibody and an actin antibody as a control for equal loading. p38 expression was suppressed significantly in cells transfected with clone p38-61 and moderately in cells transfected with the other clones compared with control vector transfected. (E) FasL expression is significantly reduced by p38siRNA. Jurkat T cells were electroporated with 5 μg plasmid-expressing vector, pNef, or p38siRNA (clone p38-61) and pNef plasmids as indicated. At 48 hours after transfection, the surface levels of FasL expression were determined by flow cytometry using a FasL-specific antibody. Filled histograms show the FasL expression, and open histograms represent isotype-matched control antibodies. p38 siRNA inhibited Nef-induced FasL induction. Similar results were obtained in 3 independent experiments. Transfection efficiency was monitored by cotransfection of a pCMV plasmid encoding GFP, which also served as a marker for gating on transfected cells. (F) p38 siRNA does not affect Nef expression. Total protein extracts were prepared from the groups transfected with vector, pNef, or p38siRNA plus pNef. Of each protein sample, 50 μg was separated on 12% SDS-PAGE and analyzed by Western blot with a polyclonal antibody against Nef antibody. Immunoblotting was also performed using an antiactin antibody as an internal control.

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