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Figure 4 | BMC Immunology

Figure 4

From: Identification of human thioredoxin as a novel IFN-gamma-induced factor: Mechanism of induction and its role in cytokine production

Figure 4

Analysis of signaling pathways activated by IFN-γ and the effects of signaling inhibitors on thioredoxin gene expression. A. Analysis of Jak, Akt, and MAPK pathways during IFN-γ signal transduction in THP1 cells. THP1 cells (1 × 106) were treated with media alone or IFN-γ under serum-free conditions for the indicated times. The total cell lysates were then prepared and analyzed by Western blot to determine the activation status of Jak1/Stat1, Akt, and MAPKs as described in the text. B. Effects of signaling inhibitors on IFN-γ-induced thioredoxin gene expression. THP1 cells (1 × 106) were pretreated with LY294002 (20 μM), PD98059 (50 μM), SP600125 (10 μM), AG490 (10 μM), PDTC (100 μM) or SB203580 (20 μM) for 1 h and then cultured for an additional 24 h in the presence or absence of IFN-γ under serum-free conditions. The total RNA was isolated and the thioredoxin mRNA levels were then analyzed by RT-PCR. C. Analysis of activation and nuclear translocation of Stat1, NF-κB, and AP-1 induced by IFN-γ in THP1 cells. Cells (5 × 106) were treated with media alone or IFN-γ (10 ng/ml) under serum-free conditions for the indicated times. The cells were then harvested and used to prepare cytoplasmic or nuclear extracts. The extracts were then subjected to Western blot to determine the activation status of Stat1, c-Jun, c-fos or NF-κB, using antibodies to anti-phosphotyrosine Stat1, anti-Stat1, c-Jun, c-fos and NF-κB (p65). As internal controls, Cox-4 and H2A were used for cytosolic and nuclear marker proteins, respectively.

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