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Bernd Rebholz, Kai Kehe, Thomas Ruzicka, Rudolf A. Rupec 

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Presentation on theme: "Bernd Rebholz, Kai Kehe, Thomas Ruzicka, Rudolf A. Rupec "— Presentation transcript:

1 Role of NF-κB/RelA and MAPK Pathways in Keratinocytes in Response to Sulfur Mustard 
Bernd Rebholz, Kai Kehe, Thomas Ruzicka, Rudolf A. Rupec  Journal of Investigative Dermatology  Volume 128, Issue 7, Pages (July 2008) DOI: /sj.jid Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Activation of the canonical NF-κB pathway by SM. Keratinocytes were stimulated with SM for the indicated times. Cell lysates were fractionated by SDS-PAGE and then analyzed by Western blotting using antibodies that detected IKK1, IKK2, IκBα, and RelA in their unphosphorylated as well as phosphorylated condition. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 The effect of SM on the activity of NF-κB in keratinocytes, and the relevance of RelA. Keratinocytes from wild-type (a) and RelA-deficient (b) mice were challenged for the indicated times (lanes 2–7) with SM. The first lane shows unstimulated cells that were used as control (Co). PMA (50ngml−1) was used as positive control. Nuclear extracts were incubated with a 32P-labeled NF-κB probe and analyzed by electrophoretic mobility-shift assay and ELISA, and specified for NF-κB subunits by ELISA. The arrow indicates the position of specific complexes. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Analysis of the effect of SM on the activation of the Raf/MEK/ERK pathway. Keratinocytes were treated for the indicated times with SM. The first lane shows unstimulated cells that were used as control (Co). Cell lysates were analyzed with antibodies against the unphosphorylated and phosphorylated forms of Raf-1, MEK1/2, ERK1/2, MSK1, and RelA after fractionation by SDS-PAGE. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Phosphorylation of p38 MAPK by SM-triggered keratinocytes. Keratinocytes were treated for the indicated times with SM. The first lane shows unstimulated cells that were used as control (Co). Protein extracts were subjected to western blotting using antibodies against the unphosphorylated and phosphorylated p38 MAPK, as well as the phosphorylated form of MKK3/6. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 The effect of SM JNK cascade. Total cellular extracts were prepared and analyzed by western blotting for the activation status of JNK1/2. The first lane shows unstimulated cells that were used as control (Co). The activation of JNK1/2 was paralleled by the activation of two JNK kinases, MKK4 and MKK7. Activation of JNK1/2 was followed by phosphorylation of ATF-2 but not c-Jun. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 SM induces an upregulation of Cxcl1 and Cxcl2 mRNA levels. Induction of Cxcl1 and Cxcl2 upon treatment with SM. Semi-quantitative reverse transcriptase-PCR for Cxcl1 and Cxcl2 was performed and β-actin was used for standardization. Inhibition of Cxcl1 and Cxcl2 induction after 1-hour preincubation with PTD-p65–P6 (lane 3), a specific inhibitor of RelA phosphorylation at Ser536. No inhibition was detected after preincubation with control peptide (PTD) (lane 4). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

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