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
Additional studies were performed to confirm the specificity of mh2er 7.9.2 binding. The first was preabsorption, for 2 h at 20°C, of the EPO-R monoclonal antibody with 100-fold molar excess of soluble EPO receptor (Genetics Institute) or nonspecific antigen (recombinant human EPO-gen; Amgen, Thousand Oaks, CA). Additionally, Western blots were probed for expression of cytokeratin to confirm that the EPO-R bands observed were not cross-reacting. The anti-cytokeratin monoclonal antibody reagent used (AE1/AE3; Dako) recognizes cytokeratins designated #16, 8, 10, 14-16, 19, and 20 and was incubated at 1:1000 for 2 h at room temperature with the immunoblots.