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. 2004 Aug;24(15):6763–6772. doi: 10.1128/MCB.24.15.6763-6772.2004

FIG. 3.

FIG. 3.

FIG. 3.

RNS-induced JNK activation occurs in Fas-dependent but TNF-R1-independent manner. (A) C10 cells were transfected with pcDNA3 or Fas 1-210 in the presence of HA-JNK1 (0.4 μg) and then exposed to 200 μM ONOO− for 2 h or 1-μg/ml M2 plus 100-ng/ml FasL (30 min). HA-JNK was immunoprecipitated, and JNK activity was assayed by in vitro kinase assay. Levels of HA-JNK1 (bottom) are included as a loading control. (B) Cells were transfected with pcDNA3 or Fas 1-210, treated with agents as described in panel A, prior to assessment of phospho-MKK4 (top) by Western blotting. (Bottom) β-Actin expression. (C) C10 cells were transfected with pcDNA3 or p60ΔCD (1.6 μg) in the presence of HA-JNK1 (0.4 μg) and then exposed to ONOO− as described above or to 10-ng/ml TNF-α (15 min) prior to assessment of JNK activity. Levels of HA-JNK1 are included as a loading control. (D) C10 cells were transfected with pcDNA3, Fas 1-210, or p60ΔCD (1.6 μg) in the presence of HA-JNK1 (0.4 μg) and then exposed to 40 ppm of NO2 for 2 h for assessment of JNK activity. Levels of HA-JNK1 are included as a loading control. (E) Lung fibroblasts derived from wild-type C57BL/6, LPR, or TNF-R1−/− mice were exposed to ONOO−, FasL + M2, or TNF-α as described above, and JNK activity was assessed in an in vitro kinase assay (top). The bottom panel represents a portion of a JNK1 Western blot as a loading control. (F) Assessment of viability of C57BL/6 or LPR lung fibroblasts exposed to 200 μM ONOO− for 2 h, using the MTT assay. Data are expressed as percent survival compared to sham controls. *, P < 0.05 (Student's t test) compared to C57BL/6-derived ONOO−-exposed fibroblasts.