TIAM1 protein, human

Fixed cells were labeled with Alexa Fluor®488-conjugated phalloidin and Hoechst 33342 (both from Invitrogen, Carlsbad, CA) to visualize F-actin and nuclei respectively. DN-Tiam1 was localized in cells using Monoclonal ANTI-FLAG® M2 mouse primary antibody (Sigma) and the Alexa Fluor® 546 goat anti-mouse IgG secondary antibody (Invitrogen). Fluorescence was observed with an epifluorescence microscope (Zeiss Axioplan 2) equipped with a digital camera (Axiocam HRm) and image acquisition software (Axiovision ver. 4.5).
To quantify the number of CLAN-positive cells (CPCs), five to eight low-power (200X) fluorescence images from each treatment group were captured. The minimum requirement for an actin structure to be counted as a CLAN was the presence of three intensely fluorescent vertices connected by three actin spokes. Representative images of what we counted as CLANs are shown in Figure 1. The number of CPCs per image, along with the total number of cells, was counted to calculate the percentage of CPCs per image. Data were pooled from 3 experiments for each treatment and represent the mean percentage of CPC ± the standard deviation (s.d.) of the mean. To determine any effects on cell spreading, the cell areas from 6–8 of the images captured from the groups treated with or without PP2 were analyzed using Axiovision software (Figure 3C). All data were pooled from 3 experiments and represent the mean cell area ± s.d. of the mean. Statistical analysis comparing the different treatment groups for CLAN formation or for cell area was performed using ANOVA (figures 3, 4 and 6) or Student's t-test (figure 5). Where pairs of treatment groups had to be compared (figures 3 and 4), ANOVA analysis was used in conjunction with the Tukey HSD test.