Fig. 6. Immunodetection and ⁴⁵Ca²⁺ binding of PCBP-25 in EGTA-extracted supernatant from infraciliary lattice (ICL) preparation. Purification and EGTA extraction (10 mM) of ICL were performed as described in the Materials and Methods. (A) Coomassie-stained SDS-PAGE and (B) immunoblots with antiserum against centrin and with monoclonal antibody 7A9. In (A) and (B), Lane 1, ICL, 40 µg; lane 2, ICL control pellet, 40 µg; lane 3, ICL EGTA pellet, 40 µg; lane 4, ICL control supernatant, 15 µg; lane 5, ICL EGTA supernatant, 15 µg; lane 6, PCBP-25, 3 µg; lane 7, PCBP-25ß, 5 µg. In (C), the ⁴⁵Ca²⁺ blot overlay assay was performed as described in the Materials and Methods at 1.4 µM free Ca²⁺ with 20 µg of ICL EGTA supernatant. Coomassie-stained SDS-PAGE (lane 1) and autoradiograph of ⁴⁵Ca²⁺ binding (lane 2). M, M_r of markers (x10³).