Fig. 4. Ectopic cyclin G2 colocalizes with PP2A/C at MTOCs/centrosomes in CHO cells.

Optical section (0.3μM) images obtained by confocal immunofluorescence microscopy are shown. Immunosignals are shown pseudo-colored in the merge fields as indicated by the font color of the single channel labels. Single channel signals are shown in black and white for better contrast. (A) Transfected cultures were treated with nocodazole for 4 h and released into drug-free medium for a 10 min pulse (top fields in each panel) or drug treated for 30 min and released for a 3 min pulse (bottom fields) of MT regrowth prior to fixation. (B) Transfected cultures were treated with nocodazole for 2 h and released into drug-free media for a 30 min pulse (top fields in each panel) or for a 3 min pulse (bottom field) of MT regrowth prior to pre-extraction and paraformaldehyde fixation. MTs were labeled with DM1A anti-α-tubulin antibody (red in A and blue in B) and centrosomes with rabbit anti-pericentrin antibody (blue in A and red in B). (C) Cells were blocked with nocodazole (left panels for 40 min; right panels for 2 h). Cells in left panels were fixed immediately; those in the right panels were washed and released in drug-free media for 3 min prior to pre-extraction. All cells were fixed in MeOH. Staining of coverslips with anti-γ tubulin and TOTO-3 revealed centrosomes (red) and DNA (blue), respectively. (D) Cells co-transfected with HA-PP2A/C and cyclin G2-GFP expression vectors were treated with nocodazole (upper panels 25 min, lower right panel 4 h) and released (upper panels 3.5 min, lower right panel 5 min) in drug-free media. Cells in the lower left panel were untreated. All cells were pre-extracted with 0.5% Triton X-100 and fixed with paraformaldehyde. MTs are probed with DM1A anti-α-tubulin (blue) and PP2A/C with sheep anti-PP2A/C (red).