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

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 1. Trx1 is present on the cell surface of HUVEC in a reduced form and limits MAC deposition. (A) Detection of Trx1 on HUVEC by flow cytometry using rabbit anti-Trx1 serum (solid line) and preimmune serum from the same animal as a negative control (broken line). A number denotes percentage of cells staining positive for Trx1, gated above the 95th percentile of the negative control. (B) Western blots of HUVEC lysates after incubation of cells with PBS or non–membrane-permeable bIAA or bNHS to label surface proteins. Lysates were blotted for Trx1 (left panel) or biotin (right panel). (C) Western blot for Trx1 after use of neutravidin beads to precipitate biotinylated proteins from HUVEC lysates after cells were incubated with either PBS or bIAA. Each experiment was performed independently three times with similar results. (D) Trx1 activity can be assessed using an insulin assay, where in the presence of DTT, Trx1 causes reduction of insulin and subsequent aggregation of the liberated insulin b-chain, measurable by absorbance at 650 nm. The addition of Trx-specific F(ab)2 (broken lines), but not nonspecific F(ab)2 (solid lines), inhibits Trx1 activity in a concentration-dependent manner. (E) HUVEC were incubated with F(ab)2 and NHS for 1 h at 37˚C and C3b deposition was assessed by flow cytometry. (F) Preincubation of HUVEC with blocking Trx-specific F (ab)2 caused an increase in zymosan-mediated MAC deposition. (A)–(D) are each representative of three independently performed experiments. Data in (E) and (F) are shown as means of three independent experiments performed in duplicates with bars indicating SD. Statistical significance of differences was calculated using one-way ANOVA with Bonferroni post- test with *p , 0.05, **p , 0.01, ***p , 0.005. (B and C) Numbers in- dicate molecular mass markers in kilodaltons.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Cytometry, Negative Control, Staining, Western Blot, Incubation, Membrane, Activity Assay, Concentration Assay, Blocking Assay

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

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 2. Trx1 inhibits complement-mediated hemolysis in an active site-dependent manner. (A) Reducing 15% SDS-PAGE of purified His- tagged Trx1 (wild-type [WT], active site mutants, and Trx80), followed by silver staining. Lower panel shows schematic of Trx1 protein with muta- tion sites. Δ1: CC/SS variant, C32 and C35 mutated to S; Δ2: Trx80 variant, stop codon inserted after K80; and Δ3: C35S variant, C35 only mutated to S. (B) Functional testing of purified Trx proteins by an insulin reduction assay. Trx proteins and insulin were mixed in the presence (broken lines) or absence (solid lines) of DTT and aggregation of the reduced insulin b-chain followed by absorbance at 650 nm. (C) Sheep RBCs opsonized with Ab were incubated with NHS in DGVB++ buffer, and lysis was measured by the absorbance of released hemoglobin in the supernatant at 490 nm. Trx proteins were added in the presence (broken lines) or absence (solid lines) of 10 mM DTT. (D) The effect of Trx proteins on lysis trig- gered via the alternative activation pathway of complement was studied as in (C), but using rabbit RBCs in Mg++EGTA buffer, which allows the activation of the alternative pathway of complement only. (A) and (B) are representative examples of three independently performed experiments. Results shown in (C) and (D) are the averages of three independent ex- periments performed in duplicate with error bars denoting SD. Statistical analysis was performed by two-way ANOVA. *p , 0.005 as compared with BSA.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: SDS Page, Silver Staining, Variant Assay, Functional Assay, Insulin Reduction Assay, Incubation, Lysis, Activation Assay

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

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 3. Trx1 inhibits activation of complement at the level of the C5 convertase. Plates were coated with aggregated human IgG, mannan, or zymosan to trigger the classical, lectin, or alternative pathways, respec- tively. Plates were then incubated with NHS with varying amounts of Trx proteins, in GVB++ buffer for the classical and lectin pathways, and Mg++

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Activation Assay, Incubation

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

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 4. Trx80 interacts with initiators and inhibitors of complement. (A) Complement proteins were coated onto 96-well plates at 5 mg/ml and probed with Trx proteins. Binding was detected using polyclonal anti-Trx, which detects both Trx1 and Trx80. *p , 0.05 and ***p , 0.005 by two-way ANOVA, compared with BSA. (B) 125I-labeled Trx proteins were added to plates coated with complement proteins (solid lines) or BSA (broken lines) and washed, and binding was detected by gamma ray emission. Specificity of binding of [125I]Trx80 was determined by the addition of excess, unlabeled Trx80 as competition during the binding incubation (open triangles). (C) The active-site substrates of Trx1 were investigated using the C35S substrate-trapping mutant of Trx1. Only C4BP and factor H were captured from serum by the C35S mutant in comparison with binding by the CC/SS active site knockout mutant. The difference in binding between C35S and CC/SS was used to determine the active-site specific interaction. **p , 0.01 and ***p , 0.005 by two-way ANOVA. All results in this figure show the averages of at least three independent experiments carried out in duplicates; error bars represent SD. fB, Factor B; fH, factor H; fP, properdin.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Binding Assay, Labeling, Incubation, Mutagenesis, Comparison, Knock-Out

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

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 6. Trx1 and Trx80 have opposing effects on anaphylatoxin production from plasma. C5a presence was assayed by chemotaxis of neutrophils through a 3-mm pore size membrane, which was fixed and stained, and numbers of cells per field of a 3100 light microscope were counted. To assess the C5a receptor dependency of chemotaxis, cells run with samples in parallel were preincubated with 100 nM C5aRA. (A) Plasma diluted to 3% in Mg++

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: Clinical Proteomics, Chemotaxis Assay, Pore Size, Membrane, Staining, Light Microscopy

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

Article Title: Truncated and full-length thioredoxin-1 have opposing activating and inhibitory properties for human complement with relevance to endothelial surfaces.

doi: 10.4049/jimmunol.1101295

Figure Lengend Snippet: FIGURE 7. Trx1 inhibits immune complex-mediated neutrophil re- cruitment in vivo. The RPA was carried out in BABLB/c mice. Treatment groups were injected i.v. with 200 mg/ml Trx1 before initiation of the RPA. After 6 h, neutrophil chemotaxis to the site of immune complex formation was assessed by flow cytometry. Representative FACS plots are shown. The data are representative of two independent experiments; four mice per group. *p , 0.05 according to t test.

Article Snippet: Human Trx1 cDNA was obtained from Origene and cloned into pET16b (Novagen) after introduction of NdeI and BamHI restriction sites by PCR.

Techniques: In Vivo, Injection, Chemotaxis Assay, Cytometry

Journal: RNA

Article Title: Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

doi: 10.1261/rna.1905910

Figure Lengend Snippet: Western blot analysis after miRNA transfections helped validate RIP-Chip results. H4 cells were first transfected with full-length cDNA plasmids: pCMV6-LDLR, pCMV6-TXNIP, and pCMV6-DBI for 24 h, and were then transfected with 25 nM of miR-107, miR-124, miR-128, miR-320 precursors, or a negative control miRNA for additional 48 h. Cells were harvested, and Western blot analyses were performed using antibodies against each expressing proteins. Anti-β-actin antibody was used to probe each blot to monitor the total protein loadings. The three miR-128 targeted proteins shown were effectively knocked down by miR-128 transfection. LDLR was also strongly reduced by miR-124 transfection. RIP-Chip did not identify LDLR as miR-124 target, however, microarray analysis of total cell RNA showed that LDLR mRNA was significant decreased upon miR-124 transfection.

Article Snippet: Full-length cDNA (all UTR-containing) cloned in pCMV6-XL5 plasmid vectors were obtained that express human low density lipoprotein receptor (LDLR, {"type":"entrez-nucleotide","attrs":{"text":"NM_000527.2","term_id":"8051613","term_text":"NM_000527.2"}} NM_000527.2 ), thioredoxin interacting protein (TXNIP, {"type":"entrez-nucleotide","attrs":{"text":"NM_006472","term_id":"928192547","term_text":"NM_006472"}} NM_006472 ), and diazepam binding inhibitor (DBI, {"type":"entrez-nucleotide","attrs":{"text":"NM_020548.4","term_id":"54262129","term_text":"NM_020548.4"}} NM_020548.4 ) (OriGene Technologies, Inc.).

Techniques: Western Blot, Transfection, RNA Immunoprecipitation - Chromatin Immunoprecipitation, Negative Control, Expressing, Microarray

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: SiRNA knockdown of ERp57, TRX, and TG2, and the effect on cellular/extracellular TG2 activity. Caco-2 cells were incubated with specific siRNAs or mock siRNA for 48 h. The efficacy of the siRNA knockdown was determined by using Western blotting. Expression of ERp57, TRX, and TG2 was normalized against the housekeeping protein GAPDH. (A–C) SiRNA treatment significantly reduced expression of (A) ERp57, (B) TRX, and (C) TG2. Incubation with mock siRNA did not affect protein expression ( n = 4). (D) Treatment with ERp57-siRNA und TRX-siRNA did not show any off-target effects on TG2 expression ( n = 3). (E) Cellular expression of ERp57, TRX, and TG2 determined by fluorometry after siRNA knockdown. Protein levels of all three target proteins were reduced by siRNA-mediated knockdown. (F) Cellular TG2 activity was determined by fluorometry after siRNA treatment. SiRNA knockdown of TRX and TG2 reduced cellular TG2 activity ( n = 5). (G) Extracellular expression of ERp57, TRX, and TG2 was investigated in unpermeabilized Caco-2 cells by fluorometry. Protein levels of all three target proteins were significantly reduced by siRNA-mediated knockdown. (H) Extracellular TG2 activity was investigated in unpermeabilized Caco-2 cells by fluorometry. SiRNA knockdown of TRX and TG2 significantly reduced extracellular TG2 activity ( n = 5). All data are shown as mean ± SD. Statistical significance was tested by using Student’s t -test with Welch correction where appropriate. * P < 0.05, ** P < 0.01, *** P < 0.001. GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: In brief, lipofectamine 2000 (1:100; 11668019; Thermo Fisher Scientific) and the corresponding siRNAs (1:50; mock siRNA, ERp57 -siRNA, TRX -siRNA, and TG2 -siRNA from Santa Cruz Biotechnology, Dallas, TX, USA) were diluted in Opti-MEM medium (Thermo Fisher Scientific).

Techniques: Knockdown, Activity Assay, Incubation, Western Blot, Expressing