Mediators of Inflammation / 2013 / Article / Fig 3

Research Article

Radical Scavenging Activity-Based and AP-1-Targeted Anti-Inflammatory Effects of Lutein in Macrophage-Like and Skin Keratinocytic Cells

Figure 3

The effect of lutein on the activation of AP-1 and its upstream signaling cascades. (a) The levels of AP-1 family proteins, p-FRA-1, c-Fos, and c-Jun in the nuclear fraction were determined by immunoblotting analyses using antibodies against phospho- or total proteins. ((b) and (c)) Phosphoprotein or total protein levels of IκBα, p38, ERK, JNK, MKK3/6, MKK4/7, TAK1, and β-actin from cell lysates were determined by immunoblotting analyses using phospho-specific or total protein antibodies. (d) An interaction between JNK and c-Fos or p38 and c-Jun was evaluated by immunoprecipitation and immunoblotting analyses. RAW264.7 cells (  cells/mL) were incubated with lutein (30 μM) in the presence or absence of LPS (1 μg/mL) for 30 min. c-Jun or c-Fos was immunoprecipitated from whole cell lysates using a specific antibody to JNK or p38, followed by immunoblotting with antibodies to c-Fos, JNK, c-Jun, and p38, as well as rabbit immunoglobulin heavy chain. (e) The kinase activity of immunoprecipitated p38 prepared from LPS-treated RAW264.7 cells was determined by measuring the level of phospho-ATF-2. The level of phosphorylated ATF-2 was measured by immunoblotting analysis. (f) The level of IL-6 mRNA from RAW264.7 cells treated with lutein (0 to 30 μM) or enzyme inhibitors (U0126 (U0), SB203580 (SB), or SP600125 (SP)) in the presence or absence of LPS (1 μg/mL) for 6 h was determined by semiquantitative RT-PCR. Relative intensity was calculated by densitometric scanning.
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