FIGURE 5.

SIRPα forms homodimers in cis on the cell surface. Cross-linked SIRPα dimers from a mixture of CHO cells that separately expressed SIRPα-EGFP and SIRPα-Myc₆ (separate transfection; STf) were compared with dimers from a culture of CHO cells that were co-expressing SIRPα-EGFP and SIRPα-Myc₆ (co-transfection; CoTf). A, immunoblots of separately transfected and co-transfected cell lysates were probed with a murine monoclonal anti-Myc antibody (9E10; left blot) and with a rabbit polyclonal anti-EGFP antibody (right blot) after cross-linking, demonstrating that the EGFP and Myc tags did not interfere with SIRPα dimerization. B, immunoprecipitation (IP) of non-treated (C) and cross-linked (X) cell lysates of separately transfected and co-transfected cells with polyclonal rabbit anti-EGFP antibody and probe (WB) with anti-Myc antibody in order to determine the presence of SIRPα dimers that contain both Myc₆ and EGFP tags (left blot). As a positive control for immunoprecipitation, proteins precipitated by the rabbit polyclonal anti-GFP antibody were also probed with the same anti-GFP antibody (right blot). Results are representative of one of three independent experiments.