Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(10)

MATERIALS AND METHODS(10)

The PCR products were mixed with 0.1 volume of gel-loading buffer and subjected to electrophoresis on a 2% NuSieve GTG low-melting point agarose (FMC Bioproducts, Rockland, ME) and 1% agarose (Promega) gel in single-strength Tris-borate-EDTA containing 3 ^g/ml ethi-dium bromide.

Restriction enzyme digestion was performed using 10 ^l of the nested-primer PCR reaction. One microliter of AvaII was added (1.0 U/^l final concentration; Promega), and the mixture was incubated for 2 h at 37°C. Both the intact material and cut material were then subjected to agarose gel electrophoresis as described above. Fragments of 140 and 57 bp were expected, if the 197-bp PCR product ultimately derived from EPO-R mRNA.

Protein Tyrosine Phosphorylation

Freshly isolated trophoblast cells from the normal term placenta or Jar cells (10 X 106 per treatment group), deprived of serum by incubation in RPMI-1640 containing only 0.5% BSA and antibiotics for 12-18 h, were used in this study. To initiate the experiments, the serum-free medium was replaced with RPMI-1640 with or without recombinant human EPO (40 U/ml; Amgen), and cells were incubated at 37°C for 2 or 5 min. Time zero controls were cells collected after preincubation in serum-free conditions. To quench experiments, flasks containing monolayer cultures of Jar cells were placed on ice, medium was aspirated, cells were rinsed with 2 ml cold PBS+0.1 mM Na3VO4, and the monolayer was then scraped into tubes and centrifuged for 1 min at 1800 X g.

This entry was posted in Human Placenta and tagged Erythropoietin Receptor, Human Placenta, Trophoblast Cells.