Journal: Arthritis research & therapy

Article Title: Stiffness and axial pain are associated with the progression of calcification in a mouse model of diffuse idiopathic skeletal hyperostosis.

doi: 10.1186/s13075-023-03053-3

Figure Lengend Snippet: Fig. 6 ENT1−/− mice show neuroplastic changes in the cervical enlargement of the spinal cord at 6.5 months of age. A–C Representative images of immunofluorescent detection and quantification within the dorsal horn of the cervical spinal cord in wild-type and ENT1−/− mice. No differences were detected between left and right dorsal horns using Mann–Whitney’s test, so data were pooled (n = three to four female, three male per genotype). To account for anatomical and size differences in the region of interest, the upper, mid, and lower regions of the cervical enlargement were analyzed independently. A region of interest was defined around each dorsal horn to analyze laminae one to four, based on greyscale density using brightfield images. The area was standardized for each region of the cervical enlargement and the raw integrated density of immunoreactivity was averaged from up to three randomly selected sections from each region per animal. Immunoreactivity was measured for A calcitonin gene-related peptide (CGRP), B glial fibrillary acidic protein (GFAP), and C ionized calcium-binding adapter molecule 1 (IBA1). Data are plotted as mean ± SEM. *P < 0.05 by Mann–Whitney test for genotype differences and Kruskal–Wallis with Dunn’s multiple comparisons test for differences between cervical regions

Article Snippet: Immunoglobulin G isotype controls for CGRP (1:750; 5–001-A, R&D systems: Minneapolis, MN), IBA1 (1:1000; 02–6102, Thermo Fisher Scientific), and GFAP (1:500; MA110,406, Thermo Fisher Scientific), as well as secondaryonly controls, were run in parallel.

Techniques: Binding Assay, MANN-WHITNEY

Journal: Science Advances

Article Title: TDP-43 safeguards the embryo genome from L1 retrotransposition

doi: 10.1126/sciadv.abq3806

Figure Lengend Snippet: ( A ) IP of endogenous L1 ORF1p in wild-type mESCs followed by WB. n.i, nonimmunized mouse [immunoglobulin G (IgG) control]; Ab, antibody only. ( B ) Immunofluorescence of wild-type mESCs shows colocalization of endogenous L1 ORF1p and MERVL Gag in 2C-like cells. Images are maximal Z projections of confocal sections. ( C ) Immunofluorescence of mouse embryos at late two-cell (2C) stage and four-cell (4C) stage. L1 ORF1p localized on the surface of the embryo with evenly scattered foci. Also see fig. S1B. Images are maximal Z projections of confocal sections. DIC, differential interference contrast microscope. ( D ) Top: Scheme of mES::TRE-3FLAG-Dux cell line construct. 3FLAG-Dux is inserted after the TRE promoter, which drives downstream gene expression upon induction by doxycycline. Bottom: MERVL Gag and L1 ORF1p are up-regulated in mES::TRE-3FLAG-Dux cell line in a doxycycline dose-dependent manner. ( E ) Immunofluorescence of mES::TRE-3FLAG-Dux cells. Images are maximal Z projections of confocal sections. Proportion of cells expressing L1 ORF1p and MERVL Gag were increased in a doxycycline dose-dependent manner.

Article Snippet: After three washes in PBS-T, the membrane was incubated in 1/5000 dilution of the peroxidase-conjugated sheep anti-mouse immunoglobulin G (IgG) secondary antibody (MP Biomedicals) for 30 min at room temperature.

Techniques: Immunofluorescence, Microscopy, Construct, Expressing

Journal: Science Advances

Article Title: TDP-43 safeguards the embryo genome from L1 retrotransposition

doi: 10.1126/sciadv.abq3806

Figure Lengend Snippet: ( A ) Silver staining of L1 ORF1p–interacting proteins co-IPed from mES::TRE-3FLAG-Dux cells after doxycycline induction. ( B ) Volcano plot showing interactome of L1 ORF1p identified by LC-MS/MS. Horizontal axis: log 2 fold change of protein signal enrichment in anti–L1 ORF1p co-IP product versus nonimmunized IgG co-IP product; vertical axis: −log 10 false discovery rate (FDR). Blue dots represent highly enriched proteins in the L1 ORF1p interactome. Proteins that were selected for further screening are labeled. ( C ) Scheme of the retrotransposition assay. The bivalent reporter plasmid [cep99-gfp-ORFeus-Mm (EF1αEF1α); see Materials and Methods] encodes a transposition-competent L1 followed by an antisense EGFP cassette interrupted by an intron. Once transcribed, the intron is spliced, and the mature mRNA containing an uninterrupted antisense EGFP cassette can be inserted into the host genome, leading to EGFP-positive cells. TSD, target site duplication. ( D ) Effects of L1 ORF1p interactors on L1 retrotransposition was examined by fluorescence-activated cell sorting (FACS) of HEK293T cells subjected to retrotransposition assay with ectopic expression of candidate proteins in (B). Overexpression of TDP-43 markedly repressed L1 retrotransposition. Negative control representing cells transfected with empty vector. ( E ) IP of L1 ORF1p followed by WB using mES::TRE-3FLAG-Dux lysate. Interaction of endogenous L1 ORF1p and TDP-43 was confirmed.

Article Snippet: After three washes in PBS-T, the membrane was incubated in 1/5000 dilution of the peroxidase-conjugated sheep anti-mouse immunoglobulin G (IgG) secondary antibody (MP Biomedicals) for 30 min at room temperature.

Techniques: Silver Staining, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Labeling, Plasmid Preparation, Fluorescence, FACS, Expressing, Over Expression, Negative Control, Transfection

Journal: Neuropathology and applied neurobiology

Article Title: Evidence of oligodendrogliosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism.

doi: 10.1111/j.1365-2990.2012.01271.x

Figure Lengend Snippet: Figure 2. Correlation analysis between oligodendrogliosis and tyrosine hydroxylase (TH) immunolabelling in the Substantia Nigra pars compacta (SNpc) and striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. (A) Myelin basic protein (MBP) immunolabelling with the avidin–biotin 3,3′-diaminobenzidine (DAB) method clearly shows the changes, compared with the control (1), occurring in oligodendrocyte (OL) morphology 72 h (2) and 2 weeks (3) after MPTP treatment, also exemplified in the camera lucida drawings. (B) Increase in number (1, 2 and 1′, 2′), mean area (3, 4 and 3′, 4′) and total area (in mm2) (5, 6 and 5′, 6′) occupied per section by the OL-immunopositive cells inversely correlate to the levels of TH immunostaining in both SNpc and striatum. These correlations refer to the number of TH-immunopositive (TH+) cells in the SNpc (1, 3, 5 and 1′, 3′ 5′) and the optical density of the TH+ fibres in the striatum (2, 4, 6 and 2′, 4′, 6′). The Pearson correlation coefficient R2 and the corresponding P-values are indicated in each correlation graph. n = 15–20.

Article Snippet: After a rinse in buffer, sections were first incubated with a biotinylated donkey anti-sheep immunoglobulin G (IgG) secondary antibody (diluted 1:500, Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA), and successively with the ABC, diluted 1:100 (ABC kit; Vectastain Elite, Vector Labs, Burlingame, CA, USA).

Techniques: Avidin-Biotin Assay, Control, Immunostaining

Journal: Neuropathology and applied neurobiology

Article Title: Evidence of oligodendrogliosis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism.

doi: 10.1111/j.1365-2990.2012.01271.x

Figure Lengend Snippet: Figure 3. Persistent oligodendrogliosis in the striatum of parkinsonian monkeys. Caudate (A) and putamen (F) in the monkey brain are put into evidence by the red boxes in the drawings at the left-hand side. On the right of each drawing, details of myelin basic protein (MBP) immunolabelling in the striatum of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, revealed by the avidin–biotin 3,3′-diaminobenzidine (DAB) method, are shown (1, 2). The toxic treatment induces an increase in the area occupied by immunopositive cell processes in both caudate (A) and putamen (F). The corresponding confocal analysis is shown in (B,B) for the caudate and in (G,G) for the putamen. A striking increase, with respect to control (B,G), in MBP immunolabelling (red) is still observed 2 years after MPTP treatment (B,G). Cell nuclei are counterstained in blue with 4′,6-diamidino-2-phenylindole (DAPI). (C,H) Quantitative analysis conducted on the confocal images reveals a significant increase in the number of OLs in both the caudate (C) and putamen (H) of parkinsonian monkeys compared with control animals. (D,E; I,J) The increase in MBP+ cells inversely correlate with the number of tyrosine hydroxylase (TH)-immunopositive (TH+) neurones counted in the Substantia Nigra pars compacta (SNpc) (D, caudate; I, putamen) and with the optical density (O.D.) values of the TH immunolabelling of the dopaminergic axons projecting to caudate (E) and putamen (J). Control group: n = 3 monkeys; parkinsonian group: n = 5 monkeys. The histograms in C and H represent the mean standard error of the mean (SEM); ***P < 0.001, calculated by the Student’s t-test. The Pearson correlation coefficient, R2, is indicated in each of the correlation graphs (D,E,I,J).

Article Snippet: After a rinse in buffer, sections were first incubated with a biotinylated donkey anti-sheep immunoglobulin G (IgG) secondary antibody (diluted 1:500, Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA), and successively with the ABC, diluted 1:100 (ABC kit; Vectastain Elite, Vector Labs, Burlingame, CA, USA).

Techniques: Control, Avidin-Biotin Assay

Journal:

Article Title: Evidence that Equine Rhinitis A Virus VP1 Is a Target of Neutralizing Antibodies and Participates Directly in Receptor Binding

doi: 10.1128/JVI.75.19.9274-9281.2001

Figure Lengend Snippet: Anti-GST-VP1 IgG inhibits the binding of ERAV virions to Vero cells. Biotinylated ERAV virions were incubated with IgG prepared from rabbits immunized with either recombinant GST-VP1 (αVP1) or whole ERAV virions (αEA) or with prebleed sera from these rabbits (Pre-VP1 or Pre-EA, respectively) before incubation with Vero cells. Virus binding was detected by flow cytometry. Panels A and B represent the results from two independent experiments. For each sample, the mean RLMFI was obtained from duplicate samples and corrected for background binding by subtraction of the mean value obtained for the negative control sample, which has no virus or IgG added (−). Final results (relative binding) are expressed as a proportion of the RLFMI obtained for the positive control virus-only sample (+).

Article Snippet: After extensive washing in PBST, the antibodies were detected with either a 1/4,000 dilution of sheep anti-rabbit immunoglobulin G (IgG) (Silenus, Melbourne, Australia) or a 1/4,000 dilution of goat anti-horse IgG (Southern Biotechnology Associates, Birmingham, Ala.), both conjugated with horseradish peroxidase.

Techniques: Binding Assay, Incubation, Recombinant, Flow Cytometry, Negative Control, Positive Control

Journal: Infection and Immunity

Article Title: Rate of Inversion of the Salmonella enterica Shufflon Regulates Expression of Invertible DNA

doi: 10.1128/iai.73.9.5568-5577.2005

Figure Lengend Snippet: FIG. 1. SDS-PAGE of a purified fusion protein containing the Rci protein of serovar Typhi. The details are in Materials and Methods. (A) Coomassie blue-stained gel; (B) immunoblot with primary anti- His6 antibody. The protein Trx-His6-Rci, of 57,565 Da (514 aa) (lanes A1 and B1) carries the Trx and His6 tags fused to Rci. The 155-aa control protein (Trx-His6) (lanes A2 and B2) is 16,689 Da in molecular size. The approximate molecular sizes of the proteins are given in kilodaltons on the left.

Article Snippet: Immunoblotting with mouse monoclonal immunoglobulin G (IgG) anti-His6 antibody (Santa Cruz Biotechnology) was performed to confirm the identities of purified proteins.

Techniques: SDS Page, Staining, Western Blot, Control

Journal: Infection and Immunity

Article Title: Rate of Inversion of the Salmonella enterica Shufflon Regulates Expression of Invertible DNA

doi: 10.1128/iai.73.9.5568-5577.2005

Figure Lengend Snippet: FIG. 7. SDS-PAGE, followed by immunoblotting with anti-His6 as primary antibody, shows that the Rci component of a fusion protein effects protein dimerization in solution. The purification of fusion proteins in which Rci was tagged either with His6 or with both His6 and GST is described in Materials and Methods, as is the detailed conduct of the pull-down experiment. His6-Rci was mixed with either GST-His6 or GST-His6-Rci, and lanes 1 and 2 show aliquots from these mixtures. After incubation, beads attached to glutathione were added to the tubes, and proteins adsorbed to the beads were viewed by SDS-PAGE (lanes 3 and 4, from the tubes of lanes 1 and 2, respectively). The GST-His6 protein does not pull down GST-His6-Rci (compare lanes 1 and 3), but GST-His6-Rci protein pulls down His6-Rci (compare lanes 2 and 4).

Article Snippet: Immunoblotting with mouse monoclonal immunoglobulin G (IgG) anti-His6 antibody (Santa Cruz Biotechnology) was performed to confirm the identities of purified proteins.

Techniques: SDS Page, Western Blot, Incubation

Journal: Infection and Immunity

Article Title: Rate of Inversion of the Salmonella enterica Shufflon Regulates Expression of Invertible DNA

doi: 10.1128/iai.73.9.5568-5577.2005

Figure Lengend Snippet: FIG. 8. Complex formation between a labeled V1 19-bp DNA frag- ment and the Trx-His6-Rci protein may be differentially inhibited by unlabeled 19-bp DNA sequences. The details of the experiment are in Materials and Methods. Unlabeled 19-bp DNA fragments were present in molar excesses of 5-, 10-, 20-, or 30-fold to labeled V1, as indicated. The gel containing the DNA-protein complexes was exposed by autoradiography. The effectiveness of competition of unlabeled material for labeled V1 is in the order V3 V1 V2 V4. This is especially clear when the densities of label in the lanes with a 30-fold excess of unlabeled challenge 19-bp sequences are examined.

Article Snippet: Immunoblotting with mouse monoclonal immunoglobulin G (IgG) anti-His6 antibody (Santa Cruz Biotechnology) was performed to confirm the identities of purified proteins.

Techniques: Labeling, Autoradiography

Journal: Infection and Immunity

Article Title: Rate of Inversion of the Salmonella enterica Shufflon Regulates Expression of Invertible DNA

doi: 10.1128/iai.73.9.5568-5577.2005

Figure Lengend Snippet: FIG. 9. Complex formation between labeled 19-bp DNA fragments and Trx-His6-Rci protein may be differentially inhibited by unlabeled 19-bp DNA sequences. The details of the experiment are in Materials and Methods. The data are derived from tests where unlabeled DNA was present in a molar excess of 30-fold to labeled DNA. Gels con- taining complexes of Trx-His6-Rci and 19-bp labeled sequences were exposed by autoradiography. The intensities of protein-DNA complex bands were measured by densitometry and compared to band densities in control lanes lacking unlabeled challenge DNA. The effectiveness of competition of unlabeled material for labeled DNA is in the order V3 V1 V2 V4, when any of labeled V1, V2, V3, or V4 are used in the experiments.

Article Snippet: Immunoblotting with mouse monoclonal immunoglobulin G (IgG) anti-His6 antibody (Santa Cruz Biotechnology) was performed to confirm the identities of purified proteins.

Techniques: Labeling, Derivative Assay, Autoradiography, Control

Journal: Glycobiology

Article Title: The ganglioside G(D2) induces the constitutive activation of c-Met in MDA-MB-231 breast cancer cells expressing the G(D3) synthase.

doi: 10.1093/glycob/cws049

Figure Lengend Snippet: Fig. 4. Colocalization of GD2 and c-Met at the plasma membrane of GD3S+ clones. Control and GD3S+ MDA-MB-231 cells were incubated with goat AF276 anti-c-Met and mouse S220-51 anti-GD2 mAbs and revealed with red-fluorescent Alexa Fluor® 647 conjugated anti-goat IgG and green-fluorescent FITC-conjugated anti-mouse IgG. The nuclei are counterstained with Hoechst 33258. Slides were observed with a LSM 710 Laser Scanning Microscope (Carl Zeiss), numerical aperture PLAN-APOCHROMAT 63× NA 1.4 with the ZEN acquisition software.

Article Snippet: Goat anti-c-Met affinity purified polyclonal immunoglobulin G (IgG) AF276 was from R&D Systems Europe (Lille, France).

Techniques: Clinical Proteomics, Membrane, Clone Assay, Control, Incubation, Laser-Scanning Microscopy, Software