Figure 5. Immunodetection of α₃, α₅, α_v, and β₁ in materials obtained after precipitation of 92–1, Mel202, FM55P, and IGR-39 cell extracts with phaseolus vulgaris agglutinin bound to agarose. One mg of the cell extracts were incubated overnight with phaseolus vulgaris agglutinin (PHA-L) immobilized on cross-linked 4% beaded agarose. Glycoproteins were released from the complexes by boiling in electrophoresis sample buffer before being subjected to 10% SDS–PAGE. Following separation, the proteins were blotted onto PVDF membrane. After being blocked the blots were incubated with one of the following antibodies specific for different integrin subunits: α₃, α₅, α_v, and β₁. Next, the membranes were incubated with the secondary antibodies either alkaline phosphatase conjugated goat anti-rabbit IgG (for α₃, α₅, α_v, integrin subunits) or alkaline phosphatase coupled goat anti-mouse IgG (for β₁ integrin subunit). Visualization of immunoreactive proteins was achieved with the use of 4-nitroblue-tetrazolium salt/5-bromo-4-chloro-3-indolylophosphate solution. Lane S shows position of molecular weight markers.