The invading cells on quadruplicate membranes were quantitated by counting across a diameter of each memebrane under 40X microscopic magnification.
Cell membrane thickness and the gray level of extracellular area, cell membrane as well as cytoplasm were quantitated using Leica QWin image analysis and image processing software (Leica Microsystems, Wetzlar, Germany).
Cells that did not migrate were removed from the top of the membrane and the migrated cells were quantitated using an acid phosphatase colorimetric assay.
Expression of nuclear proliferating cell nuclear antigen (PCNA), MIB-1 (a marker of cell proliferation), and p53 was determined with digital image analysis, and cell membrane HER-2/neu and bcl-2 were quantitated visually.
Signals on the washed membrane were visualized by autoradiography and were quantitated by densitometry analysis.
The membranes were subjected to enhanced chemiluminescence, and bands on immunoblots were quantitated by densitometry using TINA image analysis software.
The membranes were developed using enhanced chemiluminescence (GE Healthcare), and the resulting bands were quantitated using TotalLab software (TotalLab, Ltd).
In total, 2,320 proteins were quantitated, including 804 proteins previously reported to localize to the plasma membrane.
In brief, treated cells were harvested directly in buffer containing 8 M urea and 15 mM b-mecaptoethanl, protein concentrations were quantitated, 50 100 µg were electrophoresed on a reducing Tris-Tricine gel, and electroblotted to PVDF membrane.
Cell pellets were lysed, protein extracts were quantitated, loaded onto a 10%% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane.
