Characterization of rabbit monoclonal antibody UMB-2 using transfected cells.
A, Western blot analysis of the specificity of anti-CXCR4 antibodies. Membrane preparations from HEK-293 cells stably transfected to express either CCR7 or CXCR4 were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with affinity-purified 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} hybridoma supernatant at a dilution of 1∶100. Blots were developed using enhanced chemiluminescence. Note that UMB-2 detected a band of the expected molecular weight only in CXCR4- but not in CCR7-transfected cells. Two additional experiments gave similar results. Ordinate, migration of protein molecular weight markers (Mr×10−3). B, characterization of UMB-2 by immunofluorescent staining of transfected cells. HEK-293 cells expressing CCR7 or CXCR4 were either not exposed or exposed to 100 ng/ml MIP-3 or 100 ng/ml SDF-1 for 30 min, subsequently fixed and immunofluorescently stained with 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} at a dilution of 1∶100. Note that UMB-2 detected prominent immunofluorescence at the level of the plasma membrane only in CXCR4- but not in CCR7-expressing cells, and that SDF-1 exposure induced a rapid translocation of CXCR4 receptor immunostaining from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. Scale bar, 20 µm.
