Journal:

Article Title: A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site-Dependent Translation through an Interaction Downstream of the Hepatitis C Virus Polyprotein Initiation Codon

doi: 10.1128/MCB.24.18.7878-7890.2004

Figure Lengend Snippet: NSAP1 interacts specifically with the ACR in the N-terminal part of the polyprotein. (A) (i) Schematic diagrams of HCV RNA transcripts used for RNA pull-down and UV cross-linking experiments. (ii) Five adenosine residues, at the third nucleotide position of each codon, were replaced with guanosine residues; these changes maintained the same core amino acid sequence. The asterisk in construct 18-374* denotes the nucleotide changes in the ACR. (B) Effects of competitor RNAs on binding of purified NSAP1 to probe IV. The recombinant NSAP1 protein was purified to near homogeneity and then used in UV cross-linking reactions. Recombinant NSAP1 (100 ng) was incubated with RNA probe IV in the UV cross-linking experiments in the presence (lanes 2 to 9) or absence (lane 1) of competitor RNAs. The competitor RNAs consisted of the following: 50 ng of three homopolymeric RNAs (lanes 2 to 4), 2-fold (lane 5) and 20-fold (lane 6) molar excesses of unlabeled transcript IV, a 20-fold molar excess of transcript I (lane 7), and 2-fold (lane 8) and 20-fold (lane 9) molar excesses of transcript V. (C) (i) UV cross-linking experiments were performed with 100 ng of purified NSAP1 protein and with 32P-labeled RNA probes corresponding to HCV wild-type RNAs (18-402, 18-374, 18-331, and 18-402 [lanes 1 to 3 and 5, respectively]) and the mutant RNA 18-374* (lane 4). (ii) In order to investigate whether NSAP1 can form a complex with the HCV IRES, we performed RNA pull-down experiments with S10 extracts and biotinylated RNAs corresponding to various fragments of the wild-type HCV RNAs (18-402, 18-374, and 1-331 [lanes 2 to 4]) and a mutant HCV RNA (18-374* [lane 5]). Immunoblot analysis was performed with an anti-NSAP1 polyclonal antibody.

Article Snippet: The biotinylated RNAs (15 μg) were conjugated with streptavidin-agarose resin (Pierce) in the presence of incubation buffer (10 mM Tris-Cl [pH 7.5], 150 mM KCl, 1.5 mM MgCl 2 , 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.05% Nonidet P-40).

Techniques: Sequencing, Construct, Binding Assay, Purification, Recombinant, Incubation, Labeling, Mutagenesis, Western Blot

Journal:

Article Title: A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site-Dependent Translation through an Interaction Downstream of the Hepatitis C Virus Polyprotein Initiation Codon

doi: 10.1128/MCB.24.18.7878-7890.2004

Figure Lengend Snippet: Identification of a cellular protein that interacts with HCV RNA. (A) UV cross-linking experiments using 32P-labeled HCV RNA. S10 extracts of HeLa S3 cells (20 μg) were incubated with 32P-labeled HCV RNA (nt 18 to 402) (3 × 105 cpm) without competitor RNAs (lane 1). Competition experiments were performed with 32P-labeled HCV RNA (nt 18 to 402) in the presence of 50 ng of homopolymeric RNAs [poly(A), poly(C), and poly(U)] (lanes 2 to 4), or 2- and 20-fold molar excesses of unlabeled HCV RNA (nt 18 to 402) (lanes 5 and 6). (B) HCV RNA (nt 18 to 402) affinity chromatography. A biotinylated probe (15 μg) was incubated with S10 extracts (500 μg) and nonspecific tRNAs (30 μg), and then the resin-bound proteins were resolved by SDS-10% PAGE. (C) Identification of the 65-kDa protein as NSAP1. (i) Peptide mass fingerprinting via MALDI-TOF was performed as described previously (2). The peaks labeled with arrows match the estimated tryptic peptide masses of NSAP1 with an error of <50 ppm. (ii) Oligopeptides from mass fingerprinting analysis. (D) Immunoprecipitation of UV cross-linked proteins with a 32P-labeled HCV RNA probe (nt 18 to 402). After RNase treatment, the samples were precleared with protein G-agarose resin and then immunoprecipitated with 1.5 μg of rabbit polyclonal anti-NSAP1 (lane 2) or anti-GFP (lane 1) antibody. Resin-bound proteins were resolved by SDS-10% PAGE. Lane 3, 32P-labeled proteins before immunoprecipitation.

Article Snippet: The biotinylated RNAs (15 μg) were conjugated with streptavidin-agarose resin (Pierce) in the presence of incubation buffer (10 mM Tris-Cl [pH 7.5], 150 mM KCl, 1.5 mM MgCl 2 , 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.05% Nonidet P-40).

Techniques: Labeling, Incubation, Affinity Chromatography, Peptide Mass Fingerprinting, Immunoprecipitation

Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 3. Requirement of the ligand-binding domain of Notch1 for the activation and increased cell surface expression of Notch1 by transient co-transfection of TM2D3. (a, b) Activation of Notch1 with a full-length ectodomain but not those that lack EGF repeats (Notch1 LNR, LNR CC > SS, and ΔE) (Supplementary Fig. S5 online) by co-transfection of TM2D3. Cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoblotting was performed with the indicated antibodies. Notch1 LNR CC > SS and ΔE are constitutively active. (c) Inability of TM2D3 to activate Notch1 that lacks EGF repeats 11 and 12. Experiments were conducted as in a and b. (d, e) Increased Notch1 expression at cell surface by co-transfection of TM2D3 and its independence on EGF repeats 11 and 12. Cells transfected with vectors for the indicated proteins or empty vector (Vector) as a control were incubated with or without a non-membrane permeable biotinylation reagent as indicated. After fractionation with an avidin agarose, immunoblotting was performed with the indicated antibodies. (f) Cell surface expression of TM2D3. Experiments were conducted as in (d) and (e).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Ligand Binding Assay, Activation Assay, Expressing, Cotransfection, Transfection, Plasmid Preparation, Control, Western Blot, Incubation, Membrane, Fractionation, Avidin-Biotin Assay

Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 4. Physical association of Notch1 and TM2D3. (a) Physical association of TM2D3 and Notch1. 293 T cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoprecipitation (IP) and immunoblotting were performed with the indicated antibodies. (b, c) Requirement of NRR of Notch1 for physical association with TM2D3. Experiments were conducted as in (a). (d) Requirement of LNR-A of NOTCH1 for physical association with TM2D3. A schematic diagram of NOTCH1 constructs used is shown in Supplementary Fig. S5 online. Experiments were conducted as in a. (e) Requirement of C-terminal portion including transmembrane 2 domain of TM2D3 for the activation of Notch1. Experiments were conducted as in (a). (f) No dependency on any one region of TM2D3 for the physical association with Notch1. Experiments were conducted as in (b).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Construct, Activation Assay

Journal: Immunology

Article Title: Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues

doi: 10.1046/j.1365-2567.2002.01373.x

Figure Lengend Snippet: Lack of Shiga-like toxin (SLT)-binding sites in germinal centres of the lymph nodes and Peyer’s patches from ICR mice. Acetone-fixed frozen sections of normal mouse kidneys [(a), (b) and (g)], mouse draining lymph nodes 6 days after subcutaneous immunization [(c), (d) and (e)], or mouse Peyer’s patches 4 days after oral immunization [(f) and (h),], were stained with digoxigenin-labelled SLT-1B proteins (DIG-SLT-1B) plus horseradish peroxidase (HRP)-anti-DIG [(a) and (b)]. The sections were then counterstained with haematoxylin. The other sections were stained with haematoxylin and eosin (c); biotin-conjugated peanut agglutinin (PNA) plus Texas Red avidin D (d) and (f); or DIG-SLT-1B plus fluorescein isothiocyanate (FITC)-anti-DIG (e), (g) and (h).(a): Cortical regions of the kidney. Renal tubules were strongly stained with DIG-SLT-1B (arrowheads) while glomeruli were not stained (single arrow).(b): Medullary regions of the kidney. Cross-sections of collecting ducts were strongly stained with DIG-SLT-1B.(d), (e), (f) and (h): Germinal centres (arrows) were strongly stained with PNA but not with DIG-SLT-1B. Arrowheads point towards the intestinal epithelium (f) and (h).(g): Collecting ducts in kidney medulla were strongly stained with DIG-SLT-1B, as revealed by immunofluorescence. Bars represent 50 µm [(a) and (b), × 230 magnification] or 200 µm [(c) to (h), × 70 magnification].

Article Snippet: Texas Red-avidin D, HRP-avidin D, biotin-conjugated peanut agglutinin (PNA) and 3-amino-9-ethylcarbazole (AEC) substrate kit were obtained from Vector (Burlingame, CA).

Techniques: Binding Assay, Staining, Avidin-Biotin Assay, Immunofluorescence

Journal: Immunology

Article Title: Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues

doi: 10.1046/j.1365-2567.2002.01373.x

Figure Lengend Snippet: Lack of Shiga-like toxin (SLT)-binding sites on peanut agglutinin (PNA)-positive B cells from immunized ICR mouse lymph nodes. Draining lymph node cells obtained 6 days after subcutaneous immunization were stained with phycoerythrin (PE)-anti-B220 (FL-2) and biotin-PNA/PC5-streptavidin (FL-4). Cells were also treated with (a)and (b)or without(c) and (d)digoxigenin-labelled SLT-1B proteins (DIG-SLT-1B). The binding of DIG-SLT-1B was detected by fluorescein isothiocyanate (FITC)-anti-DIG (FL-1). The gated populations representing PNAbright/B220+ cells (the square in panel a) were analysed for the binding of DIG-SLT-1B (b). FL-1 signals from PNAbright/B220+-gated cells, without treatment of DIG-SLT-1B (the square in panel c), were shown as a background (d). The binding sites for SLT on RAMOS cells were demonstrated by treatment with (f) or without (e) DIG-SLT-1B, followed by incubation with FITC-anti-DIG (FL-1).

Article Snippet: Texas Red-avidin D, HRP-avidin D, biotin-conjugated peanut agglutinin (PNA) and 3-amino-9-ethylcarbazole (AEC) substrate kit were obtained from Vector (Burlingame, CA).

Techniques: Binding Assay, Staining, Incubation