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. 2002 Jan;13(1):336–347. doi: 10.1091/mbc.01-02-0302

Figure 1.

Figure 1

Activation of the Ras/MAPK pathway disrupts the actin cytoskeleton and focal contacts in NRK cells. NRK cells were transfected with cDNAs encoding various mutant forms of Ras or empty vector (pDCR). G418-resistant clones were pooled and grown onto coverslips for immunofluorescence staining, or extracts were prepared for Western blotting. (A) Whole-cell extracts from NRK/pDCR, NRK/RasV12S35, NRK/RasV12C40, and NRK/RasV12G37 cells were immunoblotted with mAb 12CA5 (recognizing the HA tag) and anti-β-actin to ensure equal loading. The same lysates were analyzed with antiphospho-MAPK (pMAPK) or antiphospho-Akt (pAkt) antibodies. (B) NRK cells grown on glass coverslips were fixed. Actin stress fibers and focal contacts were visualized by indirect immunofluorescence using phalloidin and antivinculin mAb, respectively. Bar, 20 μm. (C) NRK/RasV12S35 cells were treated with 0.1% DMSO or 50 μM PD098059 for 48h, then fixed and stained with phalloidin and antivinculin mAb. Bar, 20 μm.