Fig. 1. Detection of rPRL cleaved forms in vesicular fractions and incubation media from acini. (A) Scanning electron micrograph of acinus obtained after enzymatic dissociation of lactating mammary tissue. The 3D organisation and the morphological integrity are well preserved. Bar, 13.8 µm. (B) Electrophoresis under reducing conditions and immunoblotting analysis of rPRL molecular forms detected in tissue and in incubation medium of acini incubated in the presence of rPRL. Incubation media and acini were treated for immunoblotting as described in Materials and Methods. Immunoblotting analysis of: rPRL forms in Hanks' medium, incubated for 60 minutes at 37°C in the presence of rPRL (lane 1); vesicular content, obtained as described in Materials and Methods, of acini incubated in the presence of 5 µg/ml rPRL for 15 minutes at 20°C, washed and then chased for 5 minutes at 37°C, a time interval corresponding to the presence of PRL inside vesicles (lane 2); medium from acini incubated without exogenous rPRL (lane 3); medium from acini incubated for 60 minutes at 37°C in the presence of 5 µg/ml rPRL (lane 4). The PRL forms generated in vitro by incubating 1.5 µg/ml rPRL in citrate-phosphate buffer pH 3.2 containing bovine cathepsin D (enzyme to protein ratio 1:200) are also shown (lane 5). Positions of the molecular mass markers (kDa) are indicated on the left.