Plasmids coding for fluorescently labeled PAK isoforms were prepared as described previously [29 (link)]. MOLM-7 cells (6 × 105) were transfected using the NeonTM transfection system (1325 V, 10 ms, 3 cycles) and 0.5 µg of DNA. The transfected cells were cultured for 24 h in RPMI medium without antibiotics, incubated for 1 h on fibronectin-coated coverslips, and localization of PAK1-full and PAK2 was analyzed by fluorescence microscopy (FV-1000 confocal microscope, Olympus).
For analysis of changes in the cell-surface contact area, cells were incubated for 90 min on fibronectin-coated surface, treated for 30 min with 10 µM FRAX597, and analyzed in the interference reflection mode. The measurement was performed by means of FV-1000 confocal microscope (Olympus), using the 405 nm laser beam and focusing on the glass surface.
For analysis of changes in the cell-surface contact area, cells were incubated for 90 min on fibronectin-coated surface, treated for 30 min with 10 µM FRAX597, and analyzed in the interference reflection mode. The measurement was performed by means of FV-1000 confocal microscope (Olympus), using the 405 nm laser beam and focusing on the glass surface.
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