Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cloning, expression, and functional characterization of TL1A-Ig.

doi: 10.4049/jimmunol.1201908

Figure Lengend Snippet: FIGURE 2. Characterization of purified TL1A-Ig by SDS-PAGE, Western blot, and gel filtration. (A) Purified TL1A-Ig was quantified using an mIgG-targeted ELISA. The nonreduced (22-ME) and reduced (+2-ME) TL1A-Ig samples were separated on SDS gel and stained with Coomassie blue or analyzed by immunoblotting using Abs to mTL1A or mIgG (B) SDS-PAGE separation of reduced TL1A-Ig after enzymatic deglycosyla- tion. Protein samples were treated with glycanases and separated on SDS- PAGE followed by staining with Coomassie blue stain. Denatured TL1A- Ig was loaded in each lane. Lane 1, Untreated control; lane 2, treated with N-Glycanase only; lane 3, treated with N-Glycanase and Sialidase A; and lane 4, treated with N-Glycanase, Sialidase A, and O-Glycanase. (C and D) Gel filtration analysis and model of expected hexameric oligomerization of TL1A-Ig. Purified TL1A-Ig (100 mg) was applied on a Superdex 200 column and subjected to size exclusion chromatography. The apparent molecular mass was calculated based on the elution volumes of the standards thyroglobulin (670 kDa), aldolase (158 kDa), and OVA (44 kDa).

Article Snippet: The mTL1A cDNAwas purchased from OriGene and the extracellular domain (ECD), containing amino acids 68–252, was PCR amplified using primers that were located upstream and downstream of the cloning sites.

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, SDS-Gel, Staining, Control, Size-exclusion Chromatography

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cloning, expression, and functional characterization of TL1A-Ig.

doi: 10.4049/jimmunol.1201908

Figure Lengend Snippet: FIGURE 3. In vitro functional activity of TL1A-Ig. (A) P815 cells were stably transfected with expression vectors encoding mTNFRSF25. TNFRSF25- P815 cells were incubated with isotype mIgG1, TL1A-Ig (in culture supernatant or purified), or 4C12 and stained with fluorochrome-conjugated anti-IgG. TNFRSF25+ cells were then visualized by flow cytometry. (B) TNFRSF25-P815 cells were exposed to titrating concentrations of IgG1, TL1A-Ig, or 4C12. Cells were directly incubated with caspase substrate solution and free rhodamine 110 was determined fluorometrically. These data are representative of more than five experiments. (C) CD4+ T cells were purified from FIR mice and activated using plate-bound anti-CD3 for 4 d. Cells were harvested and incubated with isotype IgG or TL1A-Ig followed by staining with FITC anti-mouse IgG. mTL1A-Ig–bound TNFRSF25 was analyzed using flow cytometry by gating on CD4+FIR2 Tconvs and CD4+FIR+ Tregs. (D) CD4+CD252 Tconvs or (E) CD4+FIR+ Tregs were cultured in proliferation assays with indicated stimuli: anti- CD3 (2 mg/ml; 2C11), mIL-2 (10 U/ml), 4C12 (10 mg/ml), or purified TL1A-Ig (0.1 mg/ml) in triplicates for each condition. Data are representative of three independent experiments. (F) CD4+FIR+ Tregs were cultured in titrating concentration of cyclosporin A. Cultures in (D)–(F) were pulsed with [3H]thymidine for the last 6 h of 72-h incubation and incorporated isotope was measured by liquid scintillation counting. (G) For iTreg induction, FIR Tconvs were cultured with plate-bound anti-CD3, TGF-b, retinoic acid, mIL-2, and 4C12 or TL1A-Ig, and OT-II Tconvs were cultured as above plus 1:2 APCs and OVA. Cultures were analyzed for Foxp3+ cells in the CD4 gate. One representative analysis of three independent experiments is shown. ***p , 0.001 versus appropriate control. Significance was determined by one-way ANOVA with Tukey posttest (D, E). Error bars indicate mean 6 SEM. NV, Not visible.

Article Snippet: The mTL1A cDNAwas purchased from OriGene and the extracellular domain (ECD), containing amino acids 68–252, was PCR amplified using primers that were located upstream and downstream of the cloning sites.

Techniques: In Vitro, Functional Assay, Activity Assay, Stable Transfection, Transfection, Expressing, Incubation, Staining, Cytometry, Cell Culture, Concentration Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cloning, expression, and functional characterization of TL1A-Ig.

doi: 10.4049/jimmunol.1201908

Figure Lengend Snippet: FIGURE 4. mTL1A-Ig stimulates rapid proliferation of CD4+Foxp3+ Tregs in vivo. (A) The time-related serum concentrations of 4C12 and TL1A-Ig in C57BL/6 mice (n = 3–5) was determined after a single i.p injection of 100 mg 4C12 or TL1A-Ig. Protein concentration was measured in serum samples collected at indicated time points by a sandwich ELISA specific for Armenian hamster IgG or TL1A. Concentration at each time point was calculated as a percentage of the average initial serum concentration (stable agonist concentration after equilibration between blood and tissues). Unique symbol represents each 4C12 treated (solid) mouse or TL1A-Ig (hollow) mouse. (B) The kinetics and dose-dependent expansion of Tregs in peripheral blood were determined after i.p injection of 100 mg IgG (n = 3) or TL1A-Ig (n = 4) in FIR mice for 3 consecutive days as indicated by the arrows. Mice were bled daily and the percentage of peripheral Tregs relative to total CD4+ cells was determined by flow cytometry. (C) Treg expansion was monitored in the peripheral blood while administering daily i.p injections of 100 mg IgG (n = 2) or TL1A-Ig (n = 4) starting on day 0 and ending on day 20. (D) Ten million CD4+ cells were highly purified by FACS sorting from FIR mice and adoptively transferred into CD742/2 or CD42/2 mice. After 3 d (day 0), recipient mice were treated with 100 mg TL1A-Ig or IgG followed by two consecutive doses on days 1 and 2. The percentage of Foxp3+ cells was analyzed in the spleen and pooled lymph nodes on day 6. Data are representative of two independent experiments, with two or more mice per group. Statistical analysis was performed by an unpaired two-tailed Student t test (B, C). All data are means 6 SEM (B–D). *p , 0.05, **p , 0.01, ***p , 0.001 versus control.

Article Snippet: The mTL1A cDNAwas purchased from OriGene and the extracellular domain (ECD), containing amino acids 68–252, was PCR amplified using primers that were located upstream and downstream of the cloning sites.

Techniques: In Vivo, Injection, Protein Concentration, Sandwich ELISA, Concentration Assay, Cytometry, Two Tailed Test, Control

Journal: Scientific reports

Article Title: Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.

doi: 10.1038/s41598-020-64521-3

Figure Lengend Snippet: Figure 1. Derivation and characterization of cell lines lacking Atp7a, MtI and MtII genes. (a) Primary fibroblasts were isolated from the lungs of Atp7afl/Y;MtI+/+/MtII+/+ and Atp7afl/Y;MtI−/−/MtII−/− mice and then immortalized by transfection with a plasmid expressing the SV40 large T antigen (SV40 Tag) resulting in WT and MT- cells, respectively. An adenoviral vector encoding CRE recombinase was used to delete Atp7a in WT and MT- cells to obtain ATP7A- and ATP7A-/MT- cells, respectively. (b) PCR analysis of genomic DNA was used to confirm deletion of MtI and MtII genes in both the MT- and ATP7A-/MT- cell lines. Expected PCR product sizes: MtI gene (WT = 161 bp; knockout = 176 bp); MtII gene (WT = 282 bp; knockout = 299 bp). (c) Immunoblot analysis was used to confirm the loss of ATP7A protein in both ATP7A- and ATP7A-/MT- cell lines. Tubulin was detected as a loading control. Images of full-length gels and immunoblots are provided in the supplementary data.

Article Snippet: Expression plasmids for human MTI and MTII genes were purchased from Origene (RC205942 and RC202748, respectively).

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Knock-Out, Western Blot, Control

Journal: Scientific reports

Article Title: Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.

doi: 10.1038/s41598-020-64521-3

Figure Lengend Snippet: Figure 3. Relative contributions of ATP7A and metallothioneins to Cu tolerance. (a) Cu sensitivity of WT, ATP7A-, MT- and ATP7A-/MT- cells. For each cell line, 103 cells/well were seeded in 6-well plates containing basal medium, or basal medium containing 1 µM BCS with or without the indicated concentrations of CuCl2. After 6 days, cell survival was determined using the Crystal Violet assay and imaged. (b) Quantification of Crystal Violet staining. Data are expressed as percent cell survival for each cell line normalized against its growth in BCS (mean ± SEM). (c) Complementation of ATP7A-/MT- cells with plasmids encoding human cDNAs for ATP7A ( + ATP7A), MTI ( + MTI) or MTII ( + MTII). Cells were transfected with each plasmid and then selected in basal medium supplemented with 1 µM CuCl2. Equal numbers of surviving cells (103 cells/ well) were then seeded into 6-well plates containing basal medium, 1 µM BCS or 1 µM BCS plus the indicated concentrations of Cu. After 6 days, cell survival was determined using the Crystal Violet assay and imaged. (d) Quantification of Crystal Violet staining. Data are expressed as percent cell survival for each cell line normalized against its growth in BCS (mean ± SEM).

Article Snippet: Expression plasmids for human MTI and MTII genes were purchased from Origene (RC205942 and RC202748, respectively).

Techniques: Crystal Violet Assay, Staining, Transfection, Plasmid Preparation

Journal: Scientific reports

Article Title: Generation of retinal ganglion cells with functional axons from human induced pluripotent stem cells.

doi: 10.1038/srep08344

Figure Lengend Snippet: Figure 7 | Axonal transport in retinal ganglion cells. (a) Axonal transport was investigated for investigation of anterograde rapid flow employing NTRK1 and mitochondrial expression vectors. GFP was also used as a control. Plasmid vectors that carried each of these substances as well as the CMV promoter were directly introduced into the cell body using electroporation around D34. At approximately 7–10 h post-introduction, GFP expression was observed, which indicated that effective introduction of vectors by electroporation had been obtained. NTRK1 and mitochondria were identified in the cell body and in the axon, the latter location indicating anterograde rapid axonal transport. Scale bar, 80 mm. (b) A time series of axonal transport was also confirmed by the injection of Alexa-Fluo-555 conjugated cholera toxin B into retinal ganglion cell region. Cholera toxin was transported from the cell body to the peripheral area of axons by anterograde flow within approximately 2 h after injection. Scale bar, 100 mm.

Article Snippet: The NTRK1 expression vector (RG213091; ORIGENE), the pPAmCherry-Mito Vector (TaKaRa Bio), and the pcDNATM6.2/C-EmGFP Vector (Invitrogen) were electroporated into cultured cells.

Techniques: Expressing, Control, Plasmid Preparation, Electroporation, Injection

Journal: Cells

Article Title: Identification of Novel Positive Allosteric Modulators of Neurotrophin Receptors for the Treatment of Cognitive Dysfunction

doi: 10.3390/cells10081871

Figure Lengend Snippet: Mechanism of action of triazinetriones on TrkA. ( a ) Full-length TrkA with a HA-tag (TrkA-HA) fused to the C -terminus was purified through immunoprecipitation using anti-HA agarose beads. Purified TrkA-HA was incubated with DMSO (blue circles and hatched line), ACD855 (5 µM) (black triangles and solid line), or ACD856 (1 µM) (red squares and solid line) for approx. 5 min. Thereafter, ATP was added to yield the indicated concentration. Each data point is the mean ± SEM ( n = 3). The solid lines are the curve-fit using the Michaelis–Menten equation used to calculate the apparent Vmax(app) and km(app), and the dotted lines are the 95% confidence band for each curve fit. ( b – d ) Affinity labeling and streptavidin adsorption of Trka. ( b ) Western blot of streptavidin adsorbed TrkA-HA non-covalent labeled with NHS-biotinylated triazinetrione compound (lane 2) or covalent labeled by UV-crosslinking of 100 µM sulfo-SBED biotinylated compound (lane 3). Detection of immunoreactive band was performed with anti-TrkA antibody. Supernatant loaded to the left (lane 1) was used as positive control for the Western blot. Arrows on the left indicate the migration of molecular weight markers corresponding to 198, 98, and 62 kDa. ( c ) Anti-HA agarose immunoprecipitation of cross-linked or non-crosslinked sulfo-SBED compound (AC27019-SBED) from cell lysate incubated with 100 µM AC27019-SBED. Lane 1, non-UV-crosslinking, and lane 2, UV-crosslinking of sulfo-SBED labeled TrkA-HA, both detected with streptavidin-HRP. Lanes 3 and 4 are loading controls of lanes 1 and 2, respectively, were TrkA-HA was detected by immunoblotting using the anti-TrkA antibody. Both blots were part of the same gel, but the membrane was cut in two pieces, and the proteins were detected by streptavidin-HRP (left panel) or by an anti-TrkA antibody (right panel). ( d ) Streptavidin adsorption of biotinylated compound bound to TrkA-HA. Lane 1, cell lysate used for positive control of immunodetection; lane 3, cell lysate without biotinylated compound was adsorbed to streptavidin-agarose as negative control; lanes 5 and 7, cell lysates containing sulfo-SBED compound UV-crosslinked to TrkA (from two different experiments) were adsorbed to streptavidin-agarose and immunoblotted using anti-TrkA antibody. Lanes 2, 4, and 6 are empty lanes to avoid cross-contamination between lanes.

Article Snippet: Recombinant intracellular domain (ICD) of TrkA (08-186) and single site biotinylated activated (08-486-20N) or non-activated TrkA (08-486-23N) were obtained from Carna Biosciences, Odense, Denmark.

Techniques: Purification, Immunoprecipitation, Incubation, Concentration Assay, Labeling, Adsorption, Western Blot, Positive Control, Migration, Molecular Weight, Membrane, Immunodetection, Negative Control