Figure 4

Immunolocalization of Gαi1 in the 2008, 2008/13/4 and 2008/17/4 cells, before and after treatment with taxol. To immunolocalize the Gαi1 protein, the 2008, 2008/13/4 and 2008/17/4 cells were grown on tissue culture-treated slides. After 48 hr, cells were treated with either taxol or vincristine (50 nM in case of 2008 cells, and 5 μM in case of the 2008/13/4 and 2008/17/4 cells) for 4 hr and 24 hr. At the end of each time period, the cells were fixed with 4% (w/v) paraformaldehyde and processed for immunostaining. After blocking nonspecific binding sites by incubating the slides with 5% (v/v) normal goat serum, the slides were incubated with primary antibody directed against Gαi1 (1:100 rabbit polyclonal) and β-tubulin (1:200 mouse monoclonal) for 1 hr. After washing in chilled PBS (3×), the slides were incubated with FITC-conjugated anti-rabbit antibody or rhodamine-conjugated anti-mouse antibody for 30 min. At the end of the incubation, the slides were washed again in PBS and mounted in media containing anti-fade. Localization of Gαi1 and β-tubulin was accomplished using an Olympus Confocal microscope.