Fig. 6. The RIM-BP3 protein is associated with Hook1. (A) Purification and identification of proteins associated with RIM-BP3. Protein extracts of spermatids isolated from wild-type (WT) mice and RIM-BP3 knockout (KO) mice were immunoprecipitated with anti-RIM-BP3 (N) or anti-RIM-BP3 (C) antibody. The immunoprecipitated proteins were separated by SDS-PAGE and visualized by silver staining. The prominent protein specifically co-purified with RIM-BP3 was identified as Hook1 by mass spectrometric analysis. The detected Hook1 peptides are listed in the box, with their amino acid positions indicated. (B) Co-immunoprecipitation of endogenous RIM-BP3 and Hook1. The total testicular extracts from RIM-BP3 knockout (lane 3) and wild-type mice (lane 4) were immunoprecipitated with the anti-RIM-BP3 (C) antibody. The immunoprecipitates were fractionated by SDS-PAGE and blotted with anti-Hook1 antibody. Left two lanes are 0.6% of the input extracts used for immunoprecipitation. (C) Mapping of the interaction region in RIM-BP3 by yeast two-hybrid assay. RIM-BP3 full-length and its four fragments (upper panel) were fused to GAL4AD and co-transformed into yeast with the bait construct GAL4BD-Hook1. The interaction capability of each fragment was judged by the appearance of colonies on the SD minimal medium (SD-L-T-A-H) (lower panel). (D) Mapping of the interaction region in Hook1 by yeast two-hybrid assay. Full-length Hook1 and its three fragments (upper panel) were fused to GAL4BD and co-transformed into yeast with the prey construct GAL4AD-RIM-BP3. Growth of colonies on the SD minimal medium (SD-L-T-A-H) reflects positive interaction (lower panel). MT, microtubule-binding domain; O-binding, organelle-binding domain (Mendoza-Lujambio et al., 2002).