Category Archives: Human Placenta

Expression of the Erythropoietin Receptor: RESULTS(2)

Immunoreactive EPO-R was also observed in the trophoblast layer and fetoplacental vascular endothelium, as well as in cells within the villous core and basal plate of third-trimester placentas using monoclonal antibody clone mh2er 16.5.1 (Fig. 3, A and B). All negative controls generated by substituting the mouse IgG1K isotype for the primary antibody were virtually devoid of any staining (Figs. 1C, 2C, and 3C). In preabsorption studies, 3-, 10-, and 30fold molar excess of soluble EPO-R markedly diminished the immunoreactive EPO-R, whereas 30-fold molar excess soluble TNF-R1 was without effect (Fig. 4, A-C). Finally, 30-fold molar excess of cytokeratin 8/18 did not affect EPO-R immunoreactivity (Fig. 4D), whereas cytokeratin staining after preabsorption was typically reduced by 50% (data not shown).

Continue reading

Expression of the Erythropoietin Receptor: RESULTS(1)

RESULTS(1)

Detection of EPO-R Immunoreactivity

Figures 1-3 portray the expression of immunoreactive EPO-R in the human placenta and are representative of three different placentas tested at each trimester of pregnancy. In the first-trimester placentas, immunoreactive EPO-R detected by the monoclonal antibody clone mh2er 16.5.1 was expressed by villous cytotrophoblast cells, syn-cytiotrophoblast, and fetoplacental vascular endothelium, as well as other cells scattered throughout the villous core (Fig. 1, A and B). The staining was particularly intense at the membrane interface between syncytiotrophoblast and underlying cytotrophoblast.

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(11)

Trophoblast cells remained in a suspended form, and therefore the collected cells were pelleted by centrifugation for 5 min at 800 X g. Cell pellets were then resuspended in lysis buffer containing 20 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, and 1 mM N3VO4 with protease inhibitors. After incubation for 20 min at 4°C, lysates were centrifuged for 15 min at 10 000 X g, and the supernatant was stored at -80°C.

Continue reading

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.

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(9)

Nested-primer PCR reaction was then performed. The external primers were 5′-CGA-TAT-CAC-CGT-GTC-ATC-CAC-3′ and 5′ -CAG-ATC-TTC-TGC-TTC-AGA-GCC-3′ corresponding to base pairs (bp) 394-414 in exon 3 and bp 828-848 in exon 7, respectively, and producing a PCR product of 455 bp. The internal primers were 5′-GCA- CCG-AGT-GTG-TGC-TGA-GCA-3′ and 5′-GGT-CAG-CAG-CAC-CAG-GAT-GAC-3′ corresponding to bp 605625 in exon 5 and bp 781-801 in exon 7, respectively, and producing a PCR product of 197 bp. Although the external primer set yielded product in all tissues and cells examined, the nested-primer protocol improved both sensitivity and specificity. The external and internal primers were chosen to span several introns and exons such that amplification of any contaminating genomic DNA would produce products of 3202 and 280 bp, respectively. ventolin 100 mcg

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(8)

MATERIALS AND METHODS(8)

Protein molecular masses were originally estimated by visualization relative to a prestained standard (Kalioscope broad range; Bio-Rad, Richmond, CA). More precise calculation of molecular mass was then performed by also running unstained SDS-PAGE standards (Bio-Rad), staining the blots with Coomassie blue, and then measuring the relative mobility of each stained band relative to the dye front. The molecular masses of positive EPO-R bands were then interpolated from the generated standard curve. proventil inhaler

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(7)

For negative control blots, the mouse IgGi isotype was substituted for the primary antibody. After a 5-min wash in distilled H2O and two 10-min washes with TBS plus 0.05% Tween (TBST), blots were incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Promega, Madison, WI) diluted 1:7500 in TBST. The blots were again washed in distilled H2O and TBST; this was followed by a TBS wash and equilibration in alkaline phosphatase detection buffer (100 mM Tris, pH 9.5, 150 mM NaCl, 5 mM MgCl2) for 10 min. The chemiluminescent substrate reagent CDP-Star (Boehringer Mannheim), diluted 1:200 in alkaline phosphatase detection buffer, was reacted with the blots for 5 min, and the membrane was then exposed to X-OMAT (Eastman Kodak, Rochester, NY) film for signal detection. Cheap Diskus Advair

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(6)

MATERIALS AND METHODS(6)

Western Analysis

Tissue homogenates were prepared by homogenizing frozen tissues or cell pellets for 1 min on ice with a Tekmar (Cincinnati, OH) homogenizer in twice the volume of homogenizing buffer (100 mM EDTA diluted 1:2 in methanol that was combined with an equal volume of 100 mM KCl. 50 mM Tris, 50 mM NaF, 10 mM EDTA, 0.5 mM ZnCl2,1 mM dithiothreitol, pH 6.8, and a cocktail of protease inhibitors: PMSF, antipain, leupeptin, pepstatin A, chymo-statin, and soybean trypsin inhibitor). Buy Advair Diskus Online

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(5)

Preabsorption experiments were conducted with the mh2er 16.5.1 antibody from Genetics Institute. For these studies, the antibody was used at a final concentration of 3 ^g/ml after incubation overnight at 4°C with 3-, 10-, or 30-fold molar excess of soluble EPO-R (Genetics Institute) or of an irrelevant receptor, soluble tumor necrosis factor receptor 1 (TNF-R1) (R & D Systems, Minneapolis, MN). Because of the potential for nonspecific interaction of monoclonal antibodies with cytoskeletal proteins such as cytokeratin, which are abundant in the placenta, the EPO-R antibody was also incubated overnight at 4°C with a 30-fold molar excess of the most abundant cyto-keratin types in the human placenta, cytokeratins 8 and 18 (Cortex Biochem, San Leandro, CA), or an equal volume of vehicle. In parallel experiments, the monoclonal antibody for cytokeratin 18 (clone CY-90; Sigma) was preabsorbed with the cytokeratin 8/18 to confirm the binding potential of the antigen. ampicillin antibiotic

Continue reading

Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(4)

MATERIALS AND METHODS(4)

Immunocytochemistry

Two anti-EPO-R antibodies were used—a monoclonal antibody designated mh2er 16.5.1 from Genetics Institute (Cambridge, MA) directed against secreted recombinant human (rhu) EPO-R, and a sheep polyclonal antibody from Upstate Biotechnology Incorporated (Lake Placid, NY) raised against the extracellular domain of the human EPO-R. After permeabilization of the placental tissue sections or cultured cells with 0.3% Triton X-100, quenching of endogenous peroxidase with 0.6% hydrogen peroxide in methanol, and blocking with normal horse serum, the specimens were incubated with either the monoclonal or the polyclonal antibody for 1 h at room temperature. The monoclonal and polyclonal antibodies were used at concentrations of 3-30 ^g/ml and 10 ^g/ml, respectively, which proved to be optimal based on preliminary experiments. antibiotics levaquin

Continue reading