Oligomerization of the αC domains increases the amounts of αVβ3, αVβ5, and α5β1 integrins chemically cross-linked to substrate. (A) HUVECs in serum-free DMEM were plated for 2 hours on 20 μg/mL αC monomers, αC(FXIII) oligomers, or αC(tTG) oligomers; 5 × 106 adherent cells were cross-linked to the substrates with 2 mM DTSSP in PBS for 30 minutes at 4°C and extracted 4 times for 20 minutes with 0.1% SDS. The cross-linked material was recovered by treating plates for 1 hour at 37°C with a buffer containing 100 mM DTT and 0.1% SDS and then concentrated and analyzed by SDS-PAGE and immunoblotting for the β3, β1, and β5 integrin subunits. Right lanes on the gels contained 10 ng purified αVβ3, αVβ5, and α5β1 integrins. Shown is a representative of 3 experiments. Numbers below the blots refer to the relative amounts of individual integrins, as determined by densitometry and normalized to their amounts cross-linked to αC monomers. (B) Bands corresponding to cellular integrins in panelAwere compared with external standards of purified integrins and then converted to the numbers of ligand-bound integrin receptors per cell. □ indicates αC monomers; ▪, αC(FXIII) oligomers; and ▦, αC(tTG) oligomers. Results are the means ± standard deviations of 3 independent experiments.
