Fig. 6. Western blots to show that integral membrane proteins are not among those released by phospholipase C (PLC) or by washing the cells with salt/ethanol. (A) Positive and negative control western blots probed with anti-A and anti-B, anti-Ca²⁺-pump and anti-cyclic-AMP receptor antisera. m, pre-stained molecular mass markers. Lane 1, supernatant from PLC-treated pellicle; lanes 2, 4, bacterially expressed Ca²⁺ pump/glutathione-S-transferase (GST) fusion protein; lanes 3, 5, peptide from the N terminus of the cyclic AMP receptor/GST fusion protein expressed in bacteria. Lanes 1-3 were treated with anti-A and anti-B antisera, lane 4 with anti-Ca²⁺-pump antiserum and lane 5 with anti-cyclic-AMP receptor antiserum. The filled arrowhead points to surface antigen, and the open arrowhead points to 40-60 kDa proteins. The filled arrow marks the position of the Ca²⁺ pump/GST fusion protein and the open arrow to that of the N terminus of the cyclic AMP receptor/GST fusion protein. The proteins were separated on 12 % polyacrylamide gels. (B) Western blots of supernatants from sham- and PLC-treated pellicles developed with three different antibodies. m, prestained molecular mass markers. Lanes 1, 3, 5, sham-treatment supernatants; lanes 2, 4, 6, PLC treatment supernatants. Lanes 1 and 2 were treated with anti-Ca²⁺-pump (anti-cbd) antiserum, lanes 3 and 4 with anti-cyclic-AMP-receptor antiserum (cA rec) and lanes 5 and 6 with the anti-cross-reactive-determinant (anti-crd) antiserum. The filled arrowhead points to surface antigen, and the open arrowhead to 40-60 kDa proteins. The filled arrow points to where the intact Ca²⁺ pump from pellicle is expected to run (133 kDa). The open arrow points to 48 kDa, where the cyclic AMP receptor is expected to run. 6-12 % gradient polyacrylamide gels were used. (C) Western blots of salt/ethanol washes probed with three different antisera. Lane 1, anti-Ca²⁺-pump antiserum; lane 2, anti-A and anti-B antisera; lane 3, anti-cyclic-AMP-receptor antiserum. The filled arrow points to the expected molecular mass of the native Ca²⁺ pump, and the open arrow points to that of the native cyclic AMP receptor. Gradient gels from 6 to 12 % polyacrylamide were used.