5 research outputs found

    REIIBP interacts with the SMN complex in myeloma t(4;14) cells.

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    <p>A) Identification of REIIBP binding partners. REIIBP protein partners were pulled down by a tandem affinity purification approach. To isolate a protein fraction with a low level of background contaminants, the lysates from H929::REIIBP and H929 (control sample) were passed through two different chromatography columns, an anti-Flag resin and Nickel resin. The purified populations were run on SDS acrylamide gel and stained with Coomassie blue. Discrete bands were cut from the gel and analysed by mass spectrometry. The number on the right indicates the position of the respective molecular weights in KDa. The positions for the proteins identified by Mass spectrometry are shown by arrows. B) Western blot analysis on Flag IP fractions. The names of the respective antibodies used for staining are shown. Molecular weight is shown. C–D) Endogenous REIIBP interacts with the SMN complex in myeloma cells. Co-IPs of GEMIN5 (C) and SMN (D) in t(4;14) untransduced myeloma cells. GEMIN5 and SMN were immuno precipitated from H929 cell lysates using specific antibodies. An IP with normal mouse IgG antibody was used as control. The IPs were resolved in a SDS acrylamide gel and blotted for the indicated antibodies. The black star shows the position for the heavy chains of the antibodies used in the Co-IP. Please note MMSET II in H929 cells has a shorter molecular weight than canonical 150 KDa.</p

    REIIBP SET domain controls spliceosomal snRNPs abundance.

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    <p>A) Cellular snRNPs abundance. snRNPs were immunoprecipitated with anti SmB/B′ antibody, snRNAs were purified, reverse transcribed and quantified by qPCR, (compared to the level of βactin mRNA present in the input). Each analysis is an average of three independent IPs. B) SMN complex components and SmB/B′ protein levels. Total cell lysates were immunoblotted against the relative antibodies. The black arrow indicates GEMIN3 specific bands. c) snRNAs expression analysis. qRT-PCR of total RNA. Each value is an average of three independent analysis. Figures A and C are expressed relative to control (HeLa cells, dotted line). The error bars are the SEM. ** p value <0.006, * p value <0.05.</p

    List of proteins identified as co-enriched with REIIBP by mass spectrometry.

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    <p>Proteins are ordered relative to the number of mass spectra assigned to peptide sequences unique to each protein. Only proteins with at least 10 fold enrichment in spectral counts relative to control are shown. Sequence coverage denotes the percentage of total amino acid sequence covered by the peptides detected. Asterisks denote known members of the SMN complex: GEMIN4, GEMIN5, GEMIN3 (ddx20), SMN and MEP50 (wdr77).</p
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