Figure 3.

p35 colocalizes with RasGRF2 in transfected COS-7 cells and in cortical neurons. a–c, Full-length RasGRF2 was transfected into COS-7 cells and immunostained using the monoclonal anti-RasGRF2 Ab, and this was visualized using the anti-Texas Red-conjugated secondary Ab (a). Counterstaining of the nucleus was performed using Hoescht 33342 (b). Overlay of the two stains is shown in c. d–f, To examine colocalization between RasGRF2 and p35, COS-7 cells were cotransfected with p35 and RasGRF2. RasGRF2 was immunostained with Texas Red (d), and p35 was immunostained with Oregon Green (e). The overlay showing colocalization is seen in yellow (f). Scale bar, 20 μm. g–i, 7-DIC cortical neurons were immunostained for endogenous RasGRF2 and p35. RasGRF2 was immunostained using the monoclonal anti-RasGRF2 Ab and visualized with Texas Red (g). p35 was immunostained using C19 and visualized using Oregon Green (h). The overlay shows colocalization of the two proteins in yellow (i). Colocalization is visible in the cell body and along processes of the neurons. j–l, 3-DIC cortical neurons were transfected with RasGRF2. RasGRF2 was detected using the monoclonal anti-RasGRF2 Ab and visualized using Texas Red (j), endogenous p35 was detected using C19 and visualized using Oregon Green (k), and the overlay shows the colocalization of the two proteins in yellow (l), with a distribution pattern similar to the endogenous staining described above. m–o, 3-DIC cortical neurons were cotransfected with RasGRF2 and p35. RasGRF2 was detected using the monoclonal anti-RasGRF2 Ab and visualized using Texas Red (m), p35 was detected with C19 and visualized using Oregon Green (n), and the overlay shows colocalization in yellow (o). Scale bar, 20 μm.