Western blot analysis of the XG and CD99 proteins from transfected RAG clones, somatic hybrid derivatives, and erythrocytes.

Proteins from cell lysates (RAG transfectants or somatic hybrids) or erythrocyte membranes were subjected to SDS-PAGE on a 12% (wt/vol) polyacrylamide gel, transferred to nitrocellulose sheets (Schleicher and Schuell, Dassel, Germany), probed with either NBL-1 or 12E7 monoclonal antibodies (as indicated below the blots), and revealed by chemiluminescence (ECL reagent, Amersham). (A) Western blot analysis, using indicated antibodies, of lysates from single or double transfectants (names above blots; details of transfectants given in Table 1) or nontransfected RAG cells. Right-hand panel represents Western blot analysis with monoclonal antibody 12E7 after treatment of the clone XG13-h.MIC15 (CD99-expressing, RAG transfectant) with 0, 0.1 (RDE1), or 0.25 (RDE2) units of neuraminidase (as described in “Materials and methods”). Exposure times to radiographic films were 5 minutes for NBL-1 blots, 3 and 6 seconds for 12E7 blots on middle and right hand panels, respectively. (B) Western blot analysis, using indicated antibodies, of membrane lysates from erythrocytes from Xg(a−) (male, CD99-high expressor) or Xg(a+) (female) (exposure time, 1 minute). Right-hand panel represents Western blot analysis with monoclonal antibody 12E7 after treatment of Xg(a+) (female) erythrocytes with 0, 0.1 (RDE1), or 0.25 (RDE2) units of neuraminidase (as described in “Materials and methods”) (exposure time, 10 minutes). (C) Western blot analysis, using indicated antibodies, of lysates from somatic hybrids of the single transfectants h.XG66 (XG-expressing) and MIC22 (CD99-expressing) (details of transfectants given in Table 1) (exposure time, 6 minutes). The size (kd) of the detected bands was determined using rainbow-colored protein molecular weight markers.