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  • Description:
  • Affinity purification followed by mass spectroscopy identifies proteins that specifically interact with the F2 USE. (A) Silver‐stained SDS–PAGE polyacrylamide gel of protein samples derived from affinity purification with immobilized 3′ biotinylated 21‐mer RNA oligonucleotides with the F2 USE motif (USE, lanes 1 and 5), with a mutated F2 USE motif (USEmut, lane 2) or with an unrelated sequence context (Unrel., lane 3). Lanes 1–3 show protein samples eluted with up to 2000 mM NaCl (starting concentration 150 mM NaCl). Lane 4 shows the autoradiograph of a UV crosslink that highlights the size of direct interaction partners of the F2 USE (dotted arrows indicate putative direct USE‐binding proteins that could not be unequivocally assigned by the mass spectrometry data) in comparison to the band pattern of directly and indirectly interacting proteins derived from affinity purification (lane 1). Silver‐stained bands in lane 1 were cut out and subjected to mass spectrometry (protein names are only indicated at the respective size where the respective peptide score was maximal; canonical 3′ end processing factors are highlighted in yellow; p54nrb peptides of unexpected small size are highlighted in light gray; for mass spectrometry data, see also Table I). The analysis also revealed the presence of ATP citrate synthase, LRP 130, HSP 90‐ and tubulin (asterisks), which were judged to represent likely contaminants and were not included in Table I. (B) Immunoblots of eluates from affinity purifications for proteins identified by mass spectrometry (Table I). Lanes 1–3 correspond to samples as indicated in Figure 4A. Additional information on controls for unspecific RNA–protein interaction and equal loading is available in Supplementary Figure S2.
  • License:
  • Rights Managed
  • Rights Holder:
  • John Wiley & Sons, Inc.
  • License Rights Holder:
  • Copyright © 2013 Wiley Periodicals, Inc
  • Asset Type:
  • Image
  • Asset Subtype:
  • Figure
  • Image Orientation:
  • Landscape
  • Image Dimensions:
  • 728 x 437
  • Image File Size:
  • 165 KB
  • Creator:
  • Sven Danckwardt, Isabelle Kaufmann, Marc Gentzel, Konrad U Foerstner, Anne‐Susan Gantzert, Niels H Gehring, Gabriele Neu‐Yilik, Peer Bork, Walter Keller, Matthias Wilm, Matthias W Hentze, Andreas E Kulozik
  • Credit:
  • Danckwardt, S., Kaufmann, I., Gentzel, M., Foerstner, K. U., Gantzert, A.-S., Gehring, N. H., Neu‐Yilik, G., Bork, P., Keller, W., Wilm, M., Hentze, M. W., & Kulozik, A. E. (2007). Splicing factors stimulate polyadenylation via USEs at non‐canonical 3′ end formation signals. The EMBO Journal, 26(11), 2658-2669..
  • Collection:
  • Keywords:
  • Restrictions:
  • Property Release:
  • No
  • Model Release:
  • No
  • Purchasable:
  • Yes
  • Sensitive Materials:
  • No
  • Article Authors:
  • Sven Danckwardt, Isabelle Kaufmann, Marc Gentzel, Konrad U Foerstner, Anne‐Susan Gantzert, Niels H Gehring, Gabriele Neu‐Yilik, Peer Bork, Walter Keller, Matthias Wilm, Matthias W Hentze, Andreas E Kulozik
  • Article Copyright Year:
  • 2007
  • Publication Volume:
  • 26
  • Publication Issue:
  • 11
  • Publication Date:
  • 06/06/2007
  • DOI:

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