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. 2012 May 8;287(25):20769–20773. doi: 10.1074/jbc.C112.364620

FIGURE 3.

FIGURE 3.

Activated Ras-mediated phosphorylation of MAPKs in 8-oxoG-treated cells. A, MRC5 cells were exposed to 1 μm 8-oxoG and lysed at the times indicated, and phosphorylated MEK 1/2 (p-MEK1/2) and -ERK1/2 (p-ERK1/2) levels were determined (upper panels) by Western blotting. Total MEK1/2 and ERK1/2 levels served as loading controls (lower panels). B, nuclear accumulation of phosphorylated ERK1/2 in MRC5 cells in response to 8-oxoG. Right panels, DAPI-stained nuclei. Scale bars: 10 μm. C, N-Ras-dependent ERK1/2 phosphorylation in MRC5 cells. Upper panel, levels of total Ras after depletion by siRNA to H-, K-, and N-Ras, shown by Western blotting. siRNA-transfected cells were treated with 1 μm 8-oxoG for 15 min, and phosphorylated ERK1/2 and ERK1/2 levels were assessed by Western blot analysis (middle and lower panels). D, isotype-specific activation of Ras in 8-oxoG-exposed MRC5 cells. Upper panel, activation of Ras isoforms after 8-oxoG addition (15 min). Ras-GTP levels were determined in 250 μg of cell extracts by pulldown assay (15, 16). Middle panel, H-, K-, and N-Ras levels in mock- and 8-oxoG-treated cells. Lower panel, total Ras in cell extracts (25 μg/lane) is shown by pan-Ras antibody. n = 3–6.