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. Author manuscript; available in PMC: 2009 May 6.
Published in final edited form as: Exp Cell Res. 2008 Feb 2;314(7):1540–1552. doi: 10.1016/j.yexcr.2008.01.016

Figure 1. Rho activation in EGF-stimulated carcinoma cells.

Figure 1

MTLn3 cells were starved for 3 hrs and stimulated with 5 nM EGF at 37°C for the indicated times. A) Representative Western blot of a GST-RBD pull-down showing Rho activation in MTLn3 cells. The lower gel shows the amount of total Rho in cell lysates. B) Representative fluorescent micrographs of MTLn3 cells stimulated with EGF and immunostained with anti-Rho antibody. C) Immunostaining of MTLn3 cells with anti-Rho antibody pre-absorbed with GST-Rho. D) Upper panels: Time-lapse FRET imaging of MTLn3 cells expressing the RhoA biosensor, moving randomly in 5% serum. Cells were imaged for CFP and FRET every minute for 30 minutes and the FRET/CFP ratios obtained (movie S1). Lower panels: FRET/CFP ratio of MTLn3 cells expressing the RhoA biosensor and stimulated with EGF. Images were collected every 20 seconds (movie S2 and S3). E) Measurements of the mean FRET ratio (ROI of 3 μm behind the leading edge) at various times after EGF stimulation, taken from the time lapse images. Data are the mean +/− SEM from 10 different cells. Scale bar is 10 μm.