Fig. 4.

Correlation between the formation of CDRs and the magnitude of Akt phosphorylation. (A) Phase contrast and ratio [pAkt(308)/Akt] images for MEFs under different conditions. Cells were treated with or without EGF (16 nM) or PDGF (2 nM) for 3 min. After fixation, samples were stained with anti-pAkt(308) and anti-Akt antibodies. Phase contrast, pAkt(308) and Akt images were taken and then the ratio images were obtained by dividing the pAkt(308) image by the Akt image. Arrows indicate CDRs. Scale bar: 10 µm. Color bar indicates relative values of ratio intensities. (B) Quantification of ratiometric imaging in A. The relative ratio value of EGF-stimulated cells with CDRs (EGF, CDR+) was significantly higher than that of EGF-stimulated cells without CDRs (EGF, CDR−). More than 10 cells were observed for each condition. (C) Quantification of ratiometric imaging of pAkt(308) and Akt with or without nocodazole (Noco). MEFs were stimulated by EGF (16 nM) for 3 min with or without nocodazole pretreatment. After fixation, samples were stained with anti-pAkt(308) and anti-Akt antibodies. Phase, pAkt(308) and Akt images were taken. More than 15 cells were observed for each condition. One-way ANOVA was applied for the statistics; *P<0.05.