Sedimentation profiles of U1-70K (Usp101p), U1H (Usp107p), U1J (Usp108) and U1L (Usp109p) complexes

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Proteomic analysis of the U1 snRNP of reveals three essential organism-specific proteins"</p><p></p><p>Nucleic Acids Research 2007;35(5):1391-1401.</p><p>Published online 30 Jan 2007</p><p>PMCID:PMC1865046.</p><p>© 2007 The Author(s).</p> () Protein extract (5 mg) of cells expressing U1-70K-TAP, U1H-TAP and U170-HA or U1J-TAP and U1-70K-HA or U1L-TAP and U1-10K-HA were separated on a 10–30% glycerol gradient. The gradient fractions () were precipitated with IgG antibodies and separated by SDS-PAGE, immunoblotted and probed with αTAP antibodies to determine the distribution of U1-70K-TAP, U1H-TAP, U1J-TAP and U1L-TAP, as indicated. () The gradient fractions 3–8 and 10–15 of a gradient-containing U1L-TAP and U1-70K-HA were pooled bound to IgG-Sepharose. The bound material was separated on SDS-PAGE, immunoblotted and probed with IgG and HA antibodies. An aliquot (50%) of the bound material was used to isolate RNA and hybridized with a labeled U1 probe to visualize U1 snRNA as indicated. Mo, pooled gradient fractions 10–15 of a protein extract from a wild-type strain. The pooled gradient fractions 3–8 from this gradient also showed no signals (not shown). The gradient was calibrated with small (30S) and large (50S) ribosomal subunits from