Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells
Figure Lengend Snippet: ( A ) Coimmunoprecipitation of KIF17b and TB-RBP from mouse testis extracts. A silver-stained SDS/polyacrylamide gel shows total extracts from brain (lane 1) and testis (lane 2), protein A-agarose beads preclearing controls of extracts from brain (lane 3) and testis (lane 4), and immunoprecipitated cytoplasmic S100 extracts from brain (lane 5) and testis (lane 6) using affinity-purified anti-TB-RBP. Excised protein bands were sequenced by MALDI-TOF mass spectrometry. M, marker proteins. ( B ) KIF17b expression is specific to postmeiotic male germ cells. Shown is a Northern blot of RNA (10 μg) from brain (lane 1), testis (lane 2), mixed germ cells (lane 3), pachytene spermatocytes (lane 4), round spermatids (lane 5), and elongating spermatids (lane 6). Robust expression of KIF17b mRNA is seen in the postmeiotic male germ cells. ( C ) Affinity-purified anti-KIF17b recognizes TB-RBP-associated KIF17b from testis extracts. A Western blot shows protein A-agarose beads preclearing of extracts from brain (lane 1) and testis (lane 2), brain S100 cytoplasmic extract (lane 3), anti-TB-RBP immunoprecipitation of extracts from brain (lane 4) and testis (lane 5), and the enrichment of KIF17b in the TB-RBP immunoprecipitate (lane 6). ( D ) Anti-KIF17b coimmunoprecipitates TB-RBP from testis extracts. Western blot of extract immunoprecipitated with anti-KIF17b antibodies shows TB-RBP in the immunoprecipitate. Lane 1, protein A-agarose bead preclearing; lane 2, anti-KIF17b immunoprecipitate.
Article Snippet: A partial human EST clone (GenBank accession no. ) of 937 bp encoding the C terminus of a kinesin superfamily gene matching the peptide sequences derived from the MALDI-TOF mass spectrometric analysis was amplified by RT-PCR from human testis RNA (Clontech).
Techniques: Staining, Immunoprecipitation, Affinity Purification, Mass Spectrometry, Marker, Expressing, Northern Blot, Western Blot