Journal:

Article Title: Translational Regulation of Human Papillomavirus Type 16 E7 mRNA by the Peptide SEQIKA, Shared by Rabbit ? 1 -Globin and Human Cytokeratin 7

doi: 10.1128/JVI.76.14.7040-7048.2002

Figure Lengend Snippet: Effect of rabbit reticulocyte p32 on HPV16 E7 mRNA half-life, RNase A digestion, and translation. (A) Depletion of p32 promotes in vitro E7 mRNA decay. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed with p32-depleted rabbit reticulocyte lysate. (B) Quantification of mRNA half-life by densitometric analysis. ODu, optical density units. (C) The p32 component protects E7 mRNA from RNase attack. c-myc, eIF-4E, and HPV16 E7 mRNAs were incubated with (lanes 2) RNase A, (lanes 3) RNase A plus p32 eluted from the magnetic beads, or (lanes 4) BSA (control); (lanes 1) nontreated mRNA. (D) Anti-p32 PAbs promote HPV16 E7 mRNA translation in complete rabbit reticulocyte lysate. Upper part, biotinylated translation pattern obtained following electroblotting onto a polyvinylidene difluoride membrane. Lower part, the same membrane probed with anti-E7 MAb. The positions of prestained molecular size markers are indicated (in kilodaltons).

Article Snippet: Biotinylated SEQIKA, GEQIKA, SELIKA, SEQILA, and LPGALS peptides were obtained from Primm Srl, Milan, Italy.

Techniques: In Vitro, Incubation, Magnetic Beads

Journal:

Article Title: Translational Regulation of Human Papillomavirus Type 16 E7 mRNA by the Peptide SEQIKA, Shared by Rabbit ? 1 -Globin and Human Cytokeratin 7

doi: 10.1128/JVI.76.14.7040-7048.2002

Figure Lengend Snippet: Rabbit α1-globin52-57/human CK791-96 SEQIKA binds to, prevents decay of, and blocks translation of HPV16 E7 mRNA. (A) SEQIKA fragment binds to E7 mRNA. PEMSA experiments were run by incubating unlabeled c-myc, eIF-4E, or HPV16 E7 mRNA with biotinylated control peptide LPGALS or SEQIKA. Following electrophoresis, the biotinylated peptide-mRNA complex was visualized by ECL. (B) SEQIKA fragment promotes E7 mRNA stability. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed in vitro with the p32-depleted rabbit reticulocyte lysate obtained following incubation with E7 mRNA-conjugated magnetic beads plus the 6-mer peptide SEQIKA or LPGALS. (C) Quantification of mRNA half-life by densitometric analysis. ODu, optical density units. (D) SEQIKA fragment prevents HPV16 E7 mRNA translation promoted by anti-p32 PAbs in complete rabbit reticulocyte lysate. Translation reaction mixtures were preincubated with anti-p32 PAbs (50 μg) and/or peptide (10 μg) for 10 min prior to mRNA addition. Translation reactions were electrophoresed in SDS-12% PAGE. Following electroblotting onto a polyvinylidene difluoride membrane, E7 oncoprotein expression was analyzed by Western blotting with anti-E7 MAb.

Article Snippet: Biotinylated SEQIKA, GEQIKA, SELIKA, SEQILA, and LPGALS peptides were obtained from Primm Srl, Milan, Italy.

Techniques: Electrophoresis, In Vitro, Incubation, Magnetic Beads, Expressing, Western Blot

Journal:

Article Title: Translational Regulation of Human Papillomavirus Type 16 E7 mRNA by the Peptide SEQIKA, Shared by Rabbit ? 1 -Globin and Human Cytokeratin 7

doi: 10.1128/JVI.76.14.7040-7048.2002

Figure Lengend Snippet: HPV16 E7 mRNA-SEQIKA binding: definition of the consensus amino acid sequence. (A) Hemoglobins with double and triple amino acid substitutions inside the α1-globin52-57 SEQIKA motif and numbered from 1 to 7 as described in Table ​Table22 were assayed for binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments. In the controls, mRNA was incubated in binding buffer (lane c) and binding buffer plus BSA (25 μg) (lane c1). (B) Binding assays were supplemented with 50 μg of mouse anti-p32 PAbs and run as in panel A. (C) PEMSA experiments were run by incubating unlabeled HPV16 E7 mRNA with biotinylated control peptide LPGALS, binding peptide SEQIKA, and SEQIKA-derived peptides GEQIKA, SELIKA and SEQILA, each hosting a single amino acid change. The changed residues are underlined. Following electrophoresis, the biotinylated peptide-mRNA complex was visualized by ECL.

Article Snippet: Biotinylated SEQIKA, GEQIKA, SELIKA, SEQILA, and LPGALS peptides were obtained from Primm Srl, Milan, Italy.

Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Derivative Assay, Electrophoresis

Journal: Nucleic Acids Research

Article Title: Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

doi: 10.1093/nar/gkr890

Figure Lengend Snippet: Interaction of the human La protein with domain IV of the HCV IRES. ( A ) Representative EMSA experiment of domain IV RNA (20 nM) mixed with two different concentrations (0.5 and 2.0 µM) of the following proteins: full-length La(1–408), La(1–354), La(4–325), La(1–194) and La(105–325). ( B, C, D, E ) ITC experiments of the interaction of domain IV RNA with La(1–194), La(1–229), La(4–325) and La(1–408) respectively. The normalized heat of interaction for the titrations was obtained by integrating the raw data and subtracting the heat of ligand dilution into the buffer alone. The grey line represents the best fit obtained by a non-linear least-squares procedure based on an independent binding sites model. Dissociation constants ( K d ) are shown for each interaction. The binding profile observed for the full-length protein is unchanged in the mutant La(4–325), but a dramatic decrease in binding affinity is observed for mutants lacking RRM2. ( F, G, H, I ) ITC experiments of the interaction of UUUU OH RNA with La(1–194), La(1–229), La(4–325) and La(1–408). All the proteins show similar affinities and thermodynamic profiles for the interaction with this RNA, confirming that La(1–194) is sufficient for binding to short 3′ U-rich RNA sequences.

Article Snippet: The RNA oligo IV UUUU , a mutant version of the domain IV in which the first 3 nt were changed to C and the last three to U (5′-CCCCCGUGCACCAUGAGCACGAAUUUU-3′) was purchased from Primm srl (Milan, Italy).

Techniques: Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

doi: 10.1093/nar/gkr890

Figure Lengend Snippet: Thermodynamic parameters of the association of the human La protein and mutants thereof with different RNA targets in standard conditions (20 mM Tris, 100 mM KCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.25)

Article Snippet: The RNA oligo IV UUUU , a mutant version of the domain IV in which the first 3 nt were changed to C and the last three to U (5′-CCCCCGUGCACCAUGAGCACGAAUUUU-3′) was purchased from Primm srl (Milan, Italy).

Techniques:

Journal: Nucleic Acids Research

Article Title: Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

doi: 10.1093/nar/gkr890

Figure Lengend Snippet: Comparative analysis of the interaction between La and IV UUUU RNA. ( A ) Sequence and secondary structure for IV UUUU , which retains the lower stem–loop portion of domain IV while the 5′ and 3′ extensions are mutated to oligoC and oligoU, respectively. ( B and E ) ITC experiments examining the interaction of IV UUUU with La(4–325) and La(1–194) respectively. The grey line represents the best fit obtained by a non-linear least-squares procedure based on an independent binding sites model. For comparison, the interactions of domain IV ( C and F ) and UUUU OH ( D and G ) with La(4–325) and La(1–194) respectively are reported. For the interactions of La(4–325) and La(1–194) with IV UUUU , the thermodynamic signature of binding, evaluated as the relative Δ H /– T Δ S value, is identical in both cases and is closer to the UUUU OH associations .

Article Snippet: The RNA oligo IV UUUU , a mutant version of the domain IV in which the first 3 nt were changed to C and the last three to U (5′-CCCCCGUGCACCAUGAGCACGAAUUUU-3′) was purchased from Primm srl (Milan, Italy).

Techniques: Sequencing, Binding Assay, Comparison

Journal: Virology

Article Title: HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFN? production by CD4+ T cells

doi: 10.1016/j.virol.2010.08.026

Figure Lengend Snippet: C57BL/6 recipient mice (5 per group) of CFSE-labelled OT-II T cells were immunized with E2wt particles, OVA323-339 E2 particles or soluble ovalbumin (OVA) plus poly (I:C) or PBS. 3 days after immunization, mice were sacrificed and the spleen cells analysed by flow cytometry for CFSE content (A–B), or restimulated in vitro with OVA323-339 synthetic peptide for IFNγ production analysis (C–D).

Article Snippet: The OVA 257-264 and OVA 323-339 peptides from chicken ovalbumin were purchased from Primm srl.

Techniques: Flow Cytometry, In Vitro