Journal: Immunology

Article Title: Function and expression of CD1d and invariant natural killer T‐cell receptor in the cotton rat ( Sigmodon hispidus )

doi: 10.1111/imm.12532

Figure Lengend Snippet: Cotton rat (cr) CD1d presents glycolipids and binds invariant natural killer T (iNKT) T‐cell receptor (TCR). (a) CD1d dimers loaded with the glycolipid PBS57 were diluted with CD1d vehicle‐loaded dimers (DMSO) to CD1d : PBS57 concentrations of 4, 2, 1, 0·5, 0·25, 0·125 and 0 μg/ml. Monoclonal antibody (mAb) anti‐mouse CD3 was used as a positive control (4 μg/ml) and anti‐rat CD28 mAb (4 μg/ml) was added to every sample. The dilutions were coated on a 96‐well plate overnight. Cells (5 × 104) expressing a rat iNKT TCR were added and the stimulation assay was cultured for 22 hr. Supernatants were analysed by measuring mouse interleukin‐2 (IL‐2) by ELISA. The results were calculated as a percentage of the IL‐2 production following stimulation with anti‐mouse CD3. Three independent experiments were carried out in total, mean + SD were calculated with graphpad prism. (b) crCD1d dimers loaded with PBS57 (4 μg/ml, black) were used to stain cells expressing rat iNKT TCR and detected with donkey anti‐mouse secondary antibody. Dimers loaded with the vehicle DMSO (grey) served as a negative control. Geometric means are as follows: rat CD1d : DMSO 17, rat CD1d : PBS57 851, crCD1d : DMSO 4, crCD1d : PB57 1192. The staining is representative for three experiments. (c) The iNKT α‐chain 1 (pEGN crAV14 clone 1) was used to transduce mouse BW cells, together with a rat β‐chain. The transductants were stained with crCD1d dimers loaded with PBS57 (black) and detected with donkey anti‐mouse secondary antibody. Loading with the vehicle DMSO (tinted grey) served as a negative control. Geometric means are as follows: crCD1d : DMSO 3, crCD1d : PBS57 138. The staining is representative for three experiments.

Article Snippet: Supernatants were analysed with a mouse interleukin‐2 (IL‐2) sandwich ELISA (BD OptEIA™ mouse IL‐2 ELISA Kit).

Techniques: Positive Control, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Negative Control, Transduction

Journal:

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability †

doi: 10.1128/MCB.25.20.8874-8886.2005

Figure Lengend Snippet: Gene trap event in the NPM locus leads to a null NPM mutation and early embryonic lethality in mice. (A) Schematic representation of the targeting retroviral construct (middle) and the wild-type (upper) or mutated (lower) NPM alleles. Positions of oligonucleotides used for the PCR-based genotyping (see panel B) are indicated by arrows (a, b, and c). LTR: long terminal repeats; SA: splicing acceptor site; SD: splicing donor sites; β-Geo: neomycin resistance gene. (B) Left panel: PCR analysis of embryos with the indicated NPM genotypes (primer pairs used are indicated on the right). Right panel: expression of NPM protein as determined by Western blotting of lysates prepared from whole embryos. A polyclonal antibody anti-NPM (Santa Cruz) was used. (C) Wild-type (WT) and knockout (KO) NPM embryos at 10.5 dpc of embryonic development (upper panels) and immunohistochemistry analysis of NPM expression in neuroepithelial (NE) sections from the same embryos, using NPMc antibodies (lower panels). (D and E) immunohistochemistry analysis of apoptosis (D; anti-caspase-3 staining) and p53 expression levels (E; anti-p53 staining) in the developing neuroepithelium and dorsal root ganglia (DRG) of wild-type NPM and knockout 10.5 dpc embryos. (F and G) Expression of p53 target genes analyzed by quantitative PCR (p21, Pig8, Mdm2, and Bax) using mRNA from wild-type NPM (black bars), heterozygous (white bars), and knockout (gray bars) embryos. (G) Western blotting analysis of p21 protein expression in total embryo lysates.

Article Snippet: The following antisera were used: anti-BrdU (1/200; Boehringer Mannheim), anti-cleaved caspase 3 (1/200; Cell Signaling), anti-NPM (NPMc) ( 10 ), anti-H2AX (1:100; Upstate), and anti-p53 (monoclonal antibody raised against mouse p53 protein, clone AI25, kindly provided by Kristian Helin) An appropriate biotinylated secondary antibody was then applied to the slides.

Techniques: Mutagenesis, Construct, Expressing, Western Blot, Knock-Out, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal:

Article Title: Nucleophosmin Is Required for DNA Integrity and p19 Arf Protein Stability †

doi: 10.1128/MCB.25.20.8874-8886.2005

Figure Lengend Snippet: Loss of p53 gene rescues apoptosis in NPM knockout (KO) embryos. (A) p53−/− and double-knockout (p53−/− NPM−/−) embryos at different stages of development (10.5 and 12.5 dpc). (B) Analysis of apoptosis in the developing neuroepithelium (NE) of wild-type (WT), NPM−/−, p53−/− and double-knockout embryos. Immunohistochemistry analysis was performed using anti-activated caspase-3 antibody and counterstained with hematoxylin end eosin. (C) Down-regulation of NPM expression in wild-type MEFs by short interfering RNA. Cells were infected with a control (CTRL) lentivirus or a lentivirus expressing short interfering RNA for NPM (siNPM), as indicated, and analyzed by immunofluorescence using an anti-NPM antibody (NPMa) and by Western blot using anti-NPM and anti-p21 antibodies. For the growth curves, infected cells were seeded on six-well plates at a density of 104 cells per well. Cultures were harvested every day, and the number of cells was determined. The numbers refer to mean values of triplicate determinations. At day 3 of the growth curve, levels of BrdU incorporation were determined by FACS analysis (results refers to mean values of triplicate determinations). (D) Left panel: 2,000 cells derived from the yolk sac of knockout and wild-type embryos and knockout embryos reconstituted with GFP-NPM were seeded in semisolid medium supplemented with interleukin-3, interleukin-6, and stem cell factor and scored after 10 days for number of colonies. Middle panel: expression of phosphorylated p53 (anti-Ser18) and its target p21 in yolk sac-derived cells. Right panel: yolk sac-derived cells were cultured in suspension for 24 h, with (control) or without cytokines, and counted after 10 days (results are expressed as a percentage of the colonies obtained with control cells).

Article Snippet: The following antisera were used: anti-BrdU (1/200; Boehringer Mannheim), anti-cleaved caspase 3 (1/200; Cell Signaling), anti-NPM (NPMc) ( 10 ), anti-H2AX (1:100; Upstate), and anti-p53 (monoclonal antibody raised against mouse p53 protein, clone AI25, kindly provided by Kristian Helin) An appropriate biotinylated secondary antibody was then applied to the slides.

Techniques: Knock-Out, Double Knockout, Immunohistochemistry, Expressing, Small Interfering RNA, Infection, Immunofluorescence, Western Blot, BrdU Incorporation Assay, Derivative Assay, Cell Culture