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Fig. 3. Mammary acini-conditioned medium contains a PRL-cleaving activity. (A) Electrophoresis under reducing conditions and immunoblotting analysis of rPRL forms detectable in acini-conditioned medium upon incubation of rPRL. We used acini-conditioned medium, pre-heated to 100°C or not, in which 2.5 µg/ml rPRL was incubated for 60 minutes at 37°C or at 4°C. The experiment reveals the formation of 16 kDa PRL in acini-conditioned medium at 37°C, but not in those conditions (4°C, pre-heating at 100°C) that are known to abolish enzymatic activities. (B) In vitro proteolysis of PRL. Immunoblotting analysis of rPRL after incubation for 60 minutes at 37°C in citrate buffer pH 7.2 (lane 1); in citrate buffer at pH 3.2 containing 0.1 U/ml CD (lane 2); or in conditioned medium at pH 7.4 (lane 3). The lower band of the doublet (lane 3) aligns with the 16 kDa form generated by total digestion of CD at pH 3.2 (lane 2). (C) Time course (from 0 minute to 24 hours) of PRL cleavage at 37°C in acini-conditioned medium. (D) Immunoblotting analysis of rPRL forms incubated in Hanks' medium for 6 hours at 37°C in the presence of rPRL. Positions of the molecular mass markers (kDa) are indicated on the left.
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