Figure 1. Saturation isotherm of cGDPr formation in HSR membranes and Western blot analysis.

(A) HSR (60 μg) was equilibrated in buffer A for 2 min at room temperature. Subsequently, NGD was added at the concentrations indicated to initiate the formation of cGDPr. The slope of the increment in fluorescence intensity was calculated by linear regression from time intervals of 5–10 min. The insert depicts the Lineweaver–Burk transformation of the saturation isotherm. Results are means±S.E.M. of duplicates. The experiment was repeated twice with different HSR preparations and gave similar results. (B) Whole-cell lysates from Jurkat T-lymphocytes (3 μg) (lanes 1 and 3) and HSR membranes (10 μg) (lanes 2 and 4) were resolved by SDS/12% PAGE and transferred on to a nitrocellulose membrane. The proteins were stained with Ponceau S (lanes 1 and 2), and Western blot analysis was carried out as described under the Materials and methods section with a specific anti-CD38 antiserum (N-17) (lanes 3 and 4). The molecular mass standards (kDa) are indicated at the right.