Journal: Acta Neuropathologica

Article Title: Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy

doi: 10.1007/s00401-019-02034-8

Figure Lengend Snippet: Severe disruption of the cortical cytoskeleton in the Neb cKO mouse model of nemaline myopathy. Representative confocal micrographs of skeletal muscle fibres in whole-mount extensor digitorum longus muscles of control ( a , c ) and Neb cKO mice ( b , d ); tissue was immunolabelled with antibodies to β-tubulin ( a , b ) or desmin ( c , d ) and nuclei were stained with DAPI (blue). In control muscles, the skeletal muscle fibre cortex shows a regular grid-like pattern for microtubules and a striated appearance for desmin; both are largely disorganised or reduced in mutants. Note the clustering of microtubules and desmin at the periphery of nuclei, often altered or lacking in mutants. Quantifications of microtubule density ( e ), and directionality using previously developed algorithms ( f ). Immunolabelling for cytoplasmic (β- and γ-) actins, for control ( g – i ) and Neb cKO ( j – l ) mice; γ-actin ( g , j ), β-actin ( h , k ) and merged images ( i , l ). Note that microtubules, desmin and γ-actin show specific accumulation at the surface and poles of nuclei (arrowheads), which is frequently reduced or missing in mutants (asterisks). Observations were similar in three mutants and three control littermates. Graphs showing quantifications of nuclear shape parameters in muscle fibres: nuclear area ( m ), aspect ratio ( n ), circularity ( o ) and volume ( p ). Scale bars: 20 μm. Graphs, mean ± SEM, two-tailed t test. * P < 0.05), ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies were as follows (species, isotype, manufacturer, catalogue number, and dilution are given): lamin A (mouse monoclonal IgG3, Abcam, ab8980, 1:200); nesprin-1 (rabbit monoclonal IgG, Abcam, ab192234, 1:400); pericentrin (rabbit polyclonal IgG, Abcam, ab4448, 1:200); β-tubulin (clone TUB2.1, mouse monoclonal IgG1, Santa Cruz, sc-58886, 1:500); desmin (clone D33, mouse monoclonal IgG1, Dako, M076001-2, 1:400); β-actin (rabbit polyclonal IgG, Abcam, ab8227, 1:300); γ-actin (clone 2A3, mouse monoclonal IgG2b, Bio-Rad, MCA5776GA, 1:300); acetyl lys9/lys14 histone H3 (rabbit polyclonal IgG, cell signaling, #9677, 1:200).

Techniques: Disruption, Muscles, Control, Staining, Two Tailed Test

Journal: The Journal of biological chemistry

Article Title: Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation.

doi: 10.1074/jbc.M401761200

Figure Lengend Snippet: FIG. 3. Effect of group I mGluR agonist on Ca2 mobilization. Jurkat cells, resting T cells, or activated T cells were loaded with Fura-2/AM and stimulated with 100 M DHPG (lower trace) or with 5 g/ml mouse monoclonal anti-CD3 antibody as a positive control (upper trace) where indicated by an arrow. Intracellular Ca2 concentration was measured as described under “Experimental Procedures.” Repre- sentative data of one of three independent experiments are shown.

Article Snippet: Calcium peak induction was achieved by the addition of 5 g/ml mouse monoclonal anti-CD3 antibody (secreted by 33–2A3 hybridoma, kindly provided by Dr. Jaume Martorell from Hospital Clinic, Barcelona, Spain) or 100 M dihydroxyphenylglycol (DHPG) (Tocris, Bristol, UK).

Techniques: Positive Control, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation.

doi: 10.1074/jbc.M401761200

Figure Lengend Snippet: FIG. 7. Agonists of mGlu5R inhibit the anti-CD3 antibody-in- duced proliferation, but non-selective agonists suppress the in- hibition. Human T cells were incubated with medium (Control), with 100 M non-selective mGlu1/5R agonist (DHPG), or with 500 M of the mGlu5R-selective agonist (CHPG) and were immediately treated with medium or 1 ng/ml mouse monoclonal anti-CD3 antibody. Cultures were incubated for 4 days, and proliferation was determined as [3H]thy- midine incorporation. Values are expressed as proliferation index (PI), determined as [3H]thymidine incorporated in treated cells/[3H]thymi- dine incorporated in cells treated only with medium. Data are the mean S.D. of triplicates. Representative data of one of three inde- pendent experiments are shown. *, p 0.005, compared with control.

Article Snippet: Calcium peak induction was achieved by the addition of 5 g/ml mouse monoclonal anti-CD3 antibody (secreted by 33–2A3 hybridoma, kindly provided by Dr. Jaume Martorell from Hospital Clinic, Barcelona, Spain) or 100 M dihydroxyphenylglycol (DHPG) (Tocris, Bristol, UK).

Techniques: Incubation, Control

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) HuR enrichment in immunoprecipitated material from macrophages stimulated with lipopolysaccharide (LPS). The input sample could not be included due to limited availability of material. To ensure comparable protein recovery across samples, β-actin was used as a loading control. ( B ) CCL2 3′ untranslated region (3′ UTR) was detected at significant levels in the samples precipitated by anti-HuR antibody when compared to the control IgG. ( C ) CCL2 mRNA expression in anti-HuR antibody enriched immunoprecipitated material analyzed by real-time quantitative PCR (RT-qPCR) (N=4). Statistical significance was calculated using Student’s t test (*p<0.005). The error bars represent SEM. ( D ) Relative expression levels of rs13900 C and T alleles in the anti-HuR-enriched immunoprecipitated complexes obtained from macrophages stimulated with LPS (N=6). Statistical significance was calculated using Student’s t test (*p<0.005). Figure 5—source data 1. PDF file containing original western blots for supporting . Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. PDF file containing original uncropped gel for , indicating the relevant bands and treatment. Figure 5—source data 4. Original files for agarose gel displayed in . Figure 5—source data 5. Numerical data used to generate .

Article Snippet: Antibody , β-Actin (2 A3 ) (Mouse monoclonal) , Santa Cruz Biotechnology , Cat# sc-517582, RRID: AB_2833259 , WB (1:10,000).

Techniques: Immunoprecipitation, Control, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Agarose Gel Electrophoresis

Journal: eLife

Article Title: The RNA-binding protein HuR modulates the expression of the disease-linked CCL2 rs1024611G-rs13900T haplotype

doi: 10.7554/eLife.93108

Figure Lengend Snippet: ( A ) A representative western blot showing HuR protein levels in HEK-293 cells transfected with either pCMV6-HuR plasmid or HuR-targeting siRNA (HuR-siRNA). ( B ) Densitometric analysis of western blot bands. The histogram shows relative intensity of HuR bands in each sample, normalized to β-actin. The error bars represent standard error of mean obtained from three independent experiments. *p<0.025, **p<0.05. Figure 6—figure supplement 1—source data 1. PDF file containing original western blot for , indicating the relevant band and treatments. Figure 6—figure supplement 1—source data 2. Original files for western blot for .

Article Snippet: Antibody , β-Actin (2 A3 ) (Mouse monoclonal) , Santa Cruz Biotechnology , Cat# sc-517582, RRID: AB_2833259 , WB (1:10,000).

Techniques: Western Blot, Transfection, Plasmid Preparation