Journal: PLoS ONE

Article Title: Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

doi: 10.1371/journal.pone.0104513

Figure Lengend Snippet: A . Scheme of the experiment. Total RNA was annealed with 21 nt DNA oligonucleotide that was complimentary to the YB-1 mRNA sequence 150–170 nt downstream from the translation start codon, and the sample was treated with RNase H. The reaction products were separated by denaturing polyacrylamide gel electrophoresis, and YB-1 mRNA 5′-end fragments were detected by Northern blotting with a [ 32 P]-labeled DNA probe. B . Results of experiment with total RNA of human cells (HeLa and HEK293) – lanes 2 and 3, rabbit cells (reticulocytes) – lane 4, and mouse cells (NIH3T3) – lane 5. As a control (lanes 1 and 6) rabbit YB-1 mRNA (GenBank, NM_001082785.1) obtained by in vitro transcription was used. Radioautograph.

Article Snippet: The plasmid pJET 1.2 YB-1_coding region was obtained by ligation of pJET 1.2 vector (Fermentas) with PCR product amplified using the pBluescript II SK YB-1 WT construct as template and primers 5′-ATGAGCAGCGAGGCCGAG-3′ and 5′-TTACTCAGCCCCGCCCTG-3′ The template for AUC→GAC –(60–58) YB-1 mRNA in vitro synthesis was obtained using site-specific mutagenesis by overlap extension.

Techniques: Sequencing, Polyacrylamide Gel Electrophoresis, Northern Blot, Labeling, In Vitro

Journal: PLoS ONE

Article Title: Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

doi: 10.1371/journal.pone.0104513

Figure Lengend Snippet: A . Streptavidin Sepharose-immobilized biotinylated RNA fragments ( AβG mRNA, 100 nt nonspecific RNA fragment, 139 nt YB-1 mRNA 5′ UTR, 103 nt YB-1 mRNA 5′ UTR, and 136 nt YB-1 mRNA coding region fragment, 15 pmol each, were incubated with 300 µl of rabbit reticulocyte lysates. RNA-bound proteins were eluted, separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected with anti-YB-1 antibodies. Lane 1 – rabbit reticulocyte lysate as a marker, lane 2 – control experiment without (w/o) RNA, lane 3 – AβG mRNA, lane 4–100 nt nonspecific RNA fragment, lane 5–139 nt YB-1 mRNA 5′ UTR, lane 6–103 nt YB-1 mRNA 5′ UTR, lane 7–136 nt YB-1 mRNA coding region fragment. B . YB-1-specific bands from lanes 3, 5, and 6 were quantified using ImageJ software, and RNA-bound YB-1 levels were normalized to the quantity of RNA used (in moles). The amount of 103 nt 5′ UTR-bound YB-1 was taken as 100%. Values are means of three independent experiments. Errors are two standard deviations.

Article Snippet: The plasmid pJET 1.2 YB-1_coding region was obtained by ligation of pJET 1.2 vector (Fermentas) with PCR product amplified using the pBluescript II SK YB-1 WT construct as template and primers 5′-ATGAGCAGCGAGGCCGAG-3′ and 5′-TTACTCAGCCCCGCCCTG-3′ The template for AUC→GAC –(60–58) YB-1 mRNA in vitro synthesis was obtained using site-specific mutagenesis by overlap extension.

Techniques: Incubation, SDS Page, Marker, Software

Journal: PLoS ONE

Article Title: Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

doi: 10.1371/journal.pone.0104513

Figure Lengend Snippet: A . Scheme of YB-1 mRNAs with 5′UTRs of different length used in the cell-free translation system. B . 0.1 pmol of C + A + YB-1 mRNAs with 5′ UTRs of various length (139 nt – lane 2, 103 nt – lane 3, 72 nt – lane 4, 36 nt – lane 5) were translated in the rabbit reticulocyte cell-free system in the presence of [ 35 S]-Met. [ 35 S]-labeled translation products were resolved by SDS-PAGE and visualized by autoradiography. Lane 1 –translation system without exogenous mRNA. C . The relative amount of radioactivity in the bands ( B ) was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.) The level of translation of 139 5′UTR YB-1 mRNA was taken to be 100%. D . Nucleotide sequence of the 5′-terminal YB-1 mRNA fragment and its encoded amino acid sequence. An additional amino acid sequence synthesized from AUC at position –(60–58) relative to the major start codon is shown in bold. Mutation in the putative start codon is indicated. E . 0.1 pmol of C + A + YB-1 mRNAs with 140 nt WT 5′ UTR (lane 1) or 139 nt AUC→GAC (−60) 5′ UTR (lane 2) were translated in the rabbit reticulocyte cell-free system in the presence of [ 35 S]-Met. [ 35 S]-labeled translation products were resolved by SDS-PAGE and visualized by autoradiography. Lane 3 shows the translation system without exogenous mRNA.

Article Snippet: The plasmid pJET 1.2 YB-1_coding region was obtained by ligation of pJET 1.2 vector (Fermentas) with PCR product amplified using the pBluescript II SK YB-1 WT construct as template and primers 5′-ATGAGCAGCGAGGCCGAG-3′ and 5′-TTACTCAGCCCCGCCCTG-3′ The template for AUC→GAC –(60–58) YB-1 mRNA in vitro synthesis was obtained using site-specific mutagenesis by overlap extension.

Techniques: Labeling, SDS Page, Autoradiography, Radioactivity, Sequencing, Synthesized, Mutagenesis

Journal: PLoS ONE

Article Title: Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

doi: 10.1371/journal.pone.0104513

Figure Lengend Snippet: A . Scheme of predicted alternative YB-1 mRNA. B and C . Total RNAs from MCF7 (lanes 2 and 3) and HEK293 (lanes 4 and 5) were used in the reverse transcription reaction followed by PCR (lanes 2 and 4) or in PCR only (lanes 3 and 5) with primers a and b specific to intron 1 and exon 2 of the YB-1 gene ( B ) or primers c and d specific to intron 1 and exon 5 of the YB-1 gene ( C ). PCR products were resolved by electrophoresis in 2% agarose gel stained with ethidium bromide. Lane 1 shows the DNA ladder.

Article Snippet: The plasmid pJET 1.2 YB-1_coding region was obtained by ligation of pJET 1.2 vector (Fermentas) with PCR product amplified using the pBluescript II SK YB-1 WT construct as template and primers 5′-ATGAGCAGCGAGGCCGAG-3′ and 5′-TTACTCAGCCCCGCCCTG-3′ The template for AUC→GAC –(60–58) YB-1 mRNA in vitro synthesis was obtained using site-specific mutagenesis by overlap extension.

Techniques: Electrophoresis, Agarose Gel Electrophoresis, Staining

Journal: PLoS ONE

Article Title: Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

doi: 10.1371/journal.pone.0104513

Figure Lengend Snippet: MCF7 cell lysate with or without EDTA was spun through a 50% sucrose cushion at 90,000 rpm in a TLA-100 centrifuge (Beckman) for 13 min to separate postpolysomal supernatant from polysomes. Total RNA from postpolysomal supernatant and polysome fractions (resuspended pellets) was extracted with TRIzol and LiCl reprecipitated. A portion of total RNA was taken for subsequent analysis. The rest was used for reverse transcription and PCR with gene-specific primers. The total RNA samples and DNA PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromide. A . Total RNA (4 samples). B-E . DNA PCR products from: alternative form of YB-1 mRNA (primers a and b for PCR) ( B ); GAPDH mRNA ( C ); major form of YB-1 mRNA ( D ); alternative form of YB-1 mRNA (primers c and d for PCR) ( E ). Arrows indicate the expected PCR products.

Article Snippet: The plasmid pJET 1.2 YB-1_coding region was obtained by ligation of pJET 1.2 vector (Fermentas) with PCR product amplified using the pBluescript II SK YB-1 WT construct as template and primers 5′-ATGAGCAGCGAGGCCGAG-3′ and 5′-TTACTCAGCCCCGCCCTG-3′ The template for AUC→GAC –(60–58) YB-1 mRNA in vitro synthesis was obtained using site-specific mutagenesis by overlap extension.

Techniques: Agarose Gel Electrophoresis, Staining

Journal: PLoS ONE

Article Title: Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

doi: 10.1371/journal.pone.0104513

Figure Lengend Snippet: A . Scheme of long and short alternative YB-1 mRNAs. B . 0.1 pmol of C + A + YB-1 mRNAs with 140 nt WT 5′ UTR (lane 1) or long alternative YB-1 mRNA (lane 2) or short alternative YB-1 mRNA (lane 3) were translated in the rabbit reticulocyte cell-free system in the presence of [ 35 S]-Met. [ 35 S]-labeled translation products were resolved by SDS-PAGE and visualized by autoradiography. C . Scheme of mutations in the putative start and stop codons in intron 1 of alternative YB-1 mRNA. D . 0.1 pmol of C + A + long alternative YB-1 mRNA (lane 2) or long alternative YB-1 mRNA with AUG→AGA mutation (lane 3), or UAG(stop) →CAG mutation (lane 4), or AUCGUG→AUUUUG mutation (lane 5) was translated in the rabbit reticulocyte cell-free system in the presence of [ 35 S]-Met. [ 35 S]-labeled translation products were resolved by SDS-PAGE and visualized by autoradiography. Lane 1 shows the translation system without exogenous mRNA. The lower panel shows the same samples after longer electrophoresis. E . Translation reaction mixture with long alternative YB-1 mRNA or WT YB-1 mRNA was used for immunoprecipitation with preimmune antibody or YB-1 antibody. Proteins bound to antibodies were resolved by acid–urea PAGE, and [ 35 S]-labeled proteins were detected by autoradiography.

Article Snippet: The plasmid pJET 1.2 YB-1_coding region was obtained by ligation of pJET 1.2 vector (Fermentas) with PCR product amplified using the pBluescript II SK YB-1 WT construct as template and primers 5′-ATGAGCAGCGAGGCCGAG-3′ and 5′-TTACTCAGCCCCGCCCTG-3′ The template for AUC→GAC –(60–58) YB-1 mRNA in vitro synthesis was obtained using site-specific mutagenesis by overlap extension.

Techniques: Labeling, SDS Page, Autoradiography, Mutagenesis, Electrophoresis, Immunoprecipitation