Journal: The Journal of Cell Biology
Article Title: Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control
doi: 10.1083/jcb.200807033
Figure Lengend Snippet: LSm1 mRNPs contain polyadenylated mRNAs, CPEB, and CBP80. (A) Rat brain extracts were separated as before and three fractions were collected: bottom (equivalent to fractions 2–4 in Fig. 4 A), middle (fractions 5–7 in Fig. 4 A), and top (fractions 8–10 in Fig. 4 A). mRNP complexes were immunoprecipitated with mock IgGs (lanes 2, 5, and 8) or with α-LSm1 antibodies (lanes 3, 6, and 9). Lanes 1, 4, and 7 show the RT-PCR of 2.5% of the respective inputs. RNA was isolated and β-actin mRNA was detected by RT-PCR. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining; DNA markers are indicated on the left. (B) β-Actin and eEF1α1 mRNAs were amplified from a pool of the three fractions (lanes 2 and 3) or from total RNA isolated from the crude brain extract (lane 1) by the tag-addition poly(A) test (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807033/DC1). The lanes were scanned and the intensity is plotted on the right. The y axis gives the signal intensity relative to the peak of each lane. On the x axis, the position is plotted. Approximate poly(A) tail lengths were estimated from the molecular markers and the length of the amplicons without poly(A). The asterisk in the eEF1α1 panel indicates an artifact that is not dependent on tagging of the mRNA (Fig. S2). (C) Proteins were isolated from the three fractions immunopurified as in A, and the following proteins were detected by Western blotting (from top to bottom): CPEB, eIF4E, Dcp1a, CBP80, rS6, rL11, and SMN. The position of the specific bands are indicated by arrows on the right and the migration of molecular mass markers on the left; asterisks designate bands that derive from the antibodies used in the immunoprecipitations and that are therefore present in high concentration on the blot. Lanes 1, 4, and 7 show the input (10% of the immunoprecipitated material), lanes 2, 5, and 8 the mock precipitation with nonspecific IgGs, and lanes 3, 6, and 9 the specific α-LSm1 immunoprecipitations.
Article Snippet: The antibodies used in this study were as follows: affinity-purified rabbit α-LSm1 and α-LSm4 ( ); mouse SMI31 and SMI32 (Sternberger); mouse α-Calbindin (Swant); mouse α-glial fibrillary acidic protein (Sigma-Aldrich); mouse α-synaptophysin (Millipore); rabbit α-CPEB1 (gift from D. Wells, Yale University, New Haven, CT); mouse α-MAP2 (Sigma-Aldrich); α-SMN (Signal Transduction); mouse α-eIF4E (Santa Cruz Biotechnology, Inc.); goat α-orexin (Santa Cruz Biotechnology, Inc.); rabbit α-Dcp1a (gift from J. Lykke-Andersen, University of Colorado, Boulder, CO); rabbit α-rS6 and α-L11 (gift from F. Lorreni, University of Rome, Rome, Italy); and rabbit α-CBP80 (gift from I. Mattaj, European Molecular Biology Laboratory, Heidelberg, Germany).
Techniques: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Staining, Amplification, Western Blot, Migration, Concentration Assay
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