Figure 5. Immunodetection of Spam1 released from insoluble particles of epididymal luminal fluid after exposure to PI-PLC or Triton X-100. (A and B) Immunodetection in supernatant and pellet, respectively, from the initial ultracentrifugation (120 000 x g for 2 hours). In (C and D), Spam1 was analyzed in the supernatant and pellet resulting from an aliquot of the pellet in (B) after treatment with buffer alone and secondary ultracentrifugation. (E and F) Immunodetection of supernatant and pellet resulting from an aliquot of the pellet in (B) after treatment with PI-PLC and secondary ultracentrifugation. In (G and H), protein was analyzed in the supernatant and pellet resulting from an aliquot of the pellet in (B) after treatment with Triton X-100 and secondary ultracentrifugation. Only the region of the blot showing labeling is illustrated.