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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).
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