Radioimmunoblotting analysis of fractions from cells transfected with b5R cDNAs. Pellet (P) and supernatant (Sup) fractions prepared from PNS of Hela cells transfected with pCB6 (lanes 1 and 2, Vector), pCB6 containing M-cDNA (lanes 3 and 4, M) or S-cDNA (lanes 5 and 6, S) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting onto nitrocellulose. (A) Shows the blot after staining with Ponceau S; (B) shows the autoradiogram of the same blot after radioimmunostaining with anti-b5R antibodies. A total of 55 μg protein of the pellet fractions (lanes 1, 3, and 5) and 57, 60, and 78 μg protein of the supernatant fractions from cells transfected with vector alone, M-, or S-cDNA, respectively, (lanes 2, 4, and 6) were loaded. Numbers on the left indicate positions and Mr (×10−3) of molecular mass markers. The arrow in (B) indicates the position of soluble b5R. The asterisk indicates the position of a cross-reactive band in the supernatant fraction, which remains unaltered regardless of the DNA used for the transfection.
