Active Prolactin (PRL)

PRL (prolactin), also known as luteotropin, is a hormone secreted from the pituitary gland and is best known for its role in enabling mammals to produce milk. PRL plays an essential role in metabolism, regulation of the immune system. According to reports, PRL also stimulates proliferation of certain cells, including MCF-7. Thus, proliferation assay of PRL was conducted using MCF-7 cells. Briefly, MCF-7 cells were seeded into triplicate wells of 96-well plates at a density of 2,000cells/well and allowed to attach overnight, then the medium was replaced with serum-free standard DMEM prior to the addition of various concentrations of PRL. After incubated for 72h, cells were observed by inverted microscope and cell
proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly，10µL of CCK-8 solution was added to each well of the plate, then the absorbance at 450nm was measured using a microplate reader after incubating the plate for 1-4 hours at 37oC.
(A) MCF-7 cells cultured in DMEM, stimulated with 1ng/mL PRL for 72h;
(B) Unstimulated MCF-7 cells cultured in DMEM for 72h.
Figure. Cell proliferation of MCF-7 cells after stimulated with PRL.

The dose-effect curve of PRL was shown in Figure 2. It was obvious that PRL significantly promoted cell proliferation of MCF-7 cells. The ED50 for this effect is typically 3.709 to 8.973 ng/mL.
Figure. The dose-effect curve of PRL on MCF-7 cells.

PRL (prolactin), also known as luteotropin, is a hormone secreted from the pituitary gland and is best known for its role in enabling mammals to produce milk. PRL plays an essential role in metabolism, regulation of the immune system through activating its specific membrane-anchored receptor (PRLR). PRL can bind to PRLR, inducing JAK2 association that leads to downstream activation of multiple pathways that include STAT3, STAT5, PI3K, AKT, and ERK. A functional binding ELISA assay was conducted to detect the interaction of recombinant human PRL and recombinant rat JAK2. Briefly, PRL was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to JAK2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PRL pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630 nm immediately. The binding activity of recombinant human PRL and recombinant rat JAK2 was shown in Figure 1, the EC50 for this effect is 4.5 ug/mL.