Figure 3.

Adhesion to VCAM-1 (a) and CS-1 (b) of cells expressing α4 swapping mutants and the effects of Mn²⁺ (c). Wells of 96-well microtiter plates were coated with CS-1 conjugated to rat serum albumin (CS-1-RSA) at a concentration of up to 1 μg/ml in 100 μl PBS (12). For VCAM-1, wells were first coated with an anti-mouse Ck chain, and then with a VCAM-1/Ck fusion protein at a concentration of up to 1 μg/ml in 100 μl PBS (12). Clonal CHO-B2 cells expressing wild-type (wt) or mutant α4 were incubated for 1 h at 37°C with either VCAM-1 (a) or CS-1-RSA (b) immobilized to plastic wells. The adherent cells were quantified using the endogenous phosphatase assay (21). Data are expressed as means ± SD of triplicate experiments. (c) Adhesion of CHO cells to VCAM-1 was determined in the absence (filled column) and presence (hatched column) of MnCl₂ (1 mM) in the assay mixture with VCAM-1 (0.5 μg/ml coating concentration) as a substrate.