Figure 3.
Figure 3. Oxidative stress accounts for JNK up-regulation in NF-κB–deficient DCs. (A-B,D) DCs were transfected with pIRES2-EGFP or IκBαAA-pIRES2-EGFP, FACS-sorted, and cultured in the absence or presence of CD40L for 6 hours. RNA was extracted and either (A) subjected to qPCR analysis or (B,D) processed for microarray analysis. (D) Transcripts investigated included those for HMOX1, SOD1 and SOD2, FTH1, FTL, CYP-1B1, and TXN. Data are presented as x-fold induction of respective transcripts over values obtained from control-transfected, nonstimulated DCs. (A) Mean ± SD of 4 independent experiments. (C) Overexpression of XIAP or Gadd45β does not impair up-regulation of p-JNK in IκBαAA-transfected DCs. DCs were transfected with indicated vectors and stimulated for 30 minutes with CD40L. Cells were washed, fixed, and permeabilized for intracellular staining with mAbs specific for p-JNK. Data are presented as x-fold induction of JNK phosphorylation over JNK phosphorylation of nonstimulated, control-transfected cells. Mean ± SD of 3 independent experiments. (E-F) Augmented oxidative stress accounts for JNK induction in NF-κB–deficient DCs. Transfected DCs were sorted and stimulated for 30 minutes with CD40L. Cells were lysed, and proteins were derivatized for the detection of carbonylation, as described in “Materials and methods.” Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. (E) Blots were probed with DNP-specific antibodies. Strikingly augmented levels of carbonylated proteins were detected in IκBαAA-transfected DCs. Minor qualitative differences in the pattern of oxidized proteins were observed between CD40L-stimulated and nonstimulated cells. *Band of approximately 75 kDa that selectively appeared on CD40 ligation. (F) Cells preincubated for 30 minutes with N-acetyl-L-cysteine (NAC; final concentration, 60 mM) or medium only were stimulated or were not stimulated with CD40L for 30 minutes and were subjected to p-JNK immunostaining and FACS analysis. Mean ± SD of 3 independent experiments are shown.

Oxidative stress accounts for JNK up-regulation in NF-κB–deficient DCs. (A-B,D) DCs were transfected with pIRES2-EGFP or IκBαAA-pIRES2-EGFP, FACS-sorted, and cultured in the absence or presence of CD40L for 6 hours. RNA was extracted and either (A) subjected to qPCR analysis or (B,D) processed for microarray analysis. (D) Transcripts investigated included those for HMOX1, SOD1 and SOD2, FTH1, FTL, CYP-1B1, and TXN. Data are presented as x-fold induction of respective transcripts over values obtained from control-transfected, nonstimulated DCs. (A) Mean ± SD of 4 independent experiments. (C) Overexpression of XIAP or Gadd45β does not impair up-regulation of p-JNK in IκBαAA-transfected DCs. DCs were transfected with indicated vectors and stimulated for 30 minutes with CD40L. Cells were washed, fixed, and permeabilized for intracellular staining with mAbs specific for p-JNK. Data are presented as x-fold induction of JNK phosphorylation over JNK phosphorylation of nonstimulated, control-transfected cells. Mean ± SD of 3 independent experiments. (E-F) Augmented oxidative stress accounts for JNK induction in NF-κB–deficient DCs. Transfected DCs were sorted and stimulated for 30 minutes with CD40L. Cells were lysed, and proteins were derivatized for the detection of carbonylation, as described in “Materials and methods.” Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. (E) Blots were probed with DNP-specific antibodies. Strikingly augmented levels of carbonylated proteins were detected in IκBαAA-transfected DCs. Minor qualitative differences in the pattern of oxidized proteins were observed between CD40L-stimulated and nonstimulated cells. *Band of approximately 75 kDa that selectively appeared on CD40 ligation. (F) Cells preincubated for 30 minutes with N-acetyl-L-cysteine (NAC; final concentration, 60 mM) or medium only were stimulated or were not stimulated with CD40L for 30 minutes and were subjected to p-JNK immunostaining and FACS analysis. Mean ± SD of 3 independent experiments are shown.

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