Immunoprecipitation and immunoblot analysis of CLP36.

(A) Specific immunoprecipitation of platelet CLP36 by 2 different anti-CLP36 antibodies. Platelet lysates (1 mL) were incubated with anti-CLP36 Ig (lane 1), preimmune serum Ig (lane 2), or 15 μg of anti–17-peptide-antibody (lanes 4-6). The addition of 50 μg 17-peptide (lane 6) or 50 μg His-CLP36 (lane 5) to the platelet extract blocked immunoprecipitation of endogenous CLP36. Load (lanes 3 and 7) indicates aliquots of the platelet lysate before immunoprecipitation. Immunoprecipitates were separated by SDS-polyacrylamide gels and immunoblotted with anti-CLP36 Ig. Molecular mass values are given as kd; marker on the left: Rainbow full range (Amersham); marker on the right: Broad range (Biorad). (B) Expression of endogenous CLP36 in various human cell types. Homogenates (250 μg protein) of platelets, the monocytic cell line U937, human umbilical venous endothelial cells (HUVEC), human umbilical arterial endothelial cells (HUAEC), and HeLa cells were separated by SDS-PAGE, blotted, and probed with anti-CLP36 antibody.