The anti-lactadherin (P47/PaS-6/7/MfG-E8) rabbit polyclonal antibody was a gift from Dr. J.T. Rasmussen. The anti-phosphodiesterase I rabbit polyclonal (B10; nucleotide pyrophosphatase 3; E-NPP3) was a gift of Dr. M. Maurice. Anti-CD10 (neprilysin/CALLA/neutral endopeptidase) and anti-protein G-beta (against a peptide mapping at the carboxy-terminus of human G-beta 1 and recognizing G-beta 1, 2, 3, and 4) were rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-actin was a monoclonal antibody (Sigma, Saint Quentin Fallavier, France, and Prolabo, Fontenay-sous-Bois, France).
Membranes were incubated with the antisera under mild agitation overnight at 4°C or for 2 h at 37°C. The second antibody was a goat antirabbit or a goat anti-mouse antibody conjugated with peroxidase (dilution 1:5000). The peroxidase was revealed with chemoluminescent substrates and the images recorded on film or digitized with a cooled CCD camera. No reaction was observed with the secondary antibodies alone or the preimmune sera when available (for anti-CTP/HE1, -PGDS, -ERABP, and -acrosin).