Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 1. Flow cytometry sorting of the combinatorial Scl library. (A) Library expression was monitored by staining with a phycoerythrin- conjugated antibody binding to a primary anti-c-Myc antibody. The purple rectangle gate indicates cells with the highest expression. (B) The library was incubated with 5 nM LRP4, and the low-affinity library fraction was collected (purple triangle). (C) The low-affinity library was incubated with 650 nM LRP6, and the purple rectangle represents clones that bind to LRP6.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Clone Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 2. Identification of affinity-reducing mutations. Heat maps demonstrating significantly enriched Scl variants in (A) LRP4LOW library compared to SclNAIVE library fractions; (B) LRP4LOWLRP6 library compared to LRP4LOW library fractions; (C) LRP4LOW library compared to SclNAIVE library fractions that overlap with the LRP4LOWLRP6 library. The heat maps present the log2 transformation of the ER (red scale bar on the right-hand side) and highlight single mutations that significantly (A) reduce the binding affinity to LRP4, (B) reduce the binding affinity to LRP4 and retain binding to LRP6, and (C) overlap in (A) and (B). The substituting amino acids are shown on the X-axis, and the substituted positions are shown on the Y-axis. Statistical significance was determined by a two-sided Poisson exact test and multi-test corrected by the Benjamini–Hochberg FDR.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Transformation Assay, Binding Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 4. YSD binding of SclWT and the selected single-mutation variants to LRP4. Geometric mean fluorescence intensity (Geo MFI) is presented as a fold change. Recombinant yeast cells expressing SclWT or its variants were incubated with (A) 1 nM, (B) 10 nM, or (C) 50 nM soluble LRP4. The binding signal of each Scl variant was normalized first to the expression signal of the corresponding variant and then to the binding signal of SclWT at the respective LRP4 concentration. Each experiment was repeated at least three times, and the results are presented as means SD. Statistical significance was assessed using an unpaired, two-tailed Student’s t-test. *P ≤0.05; **P ≤0.01; ***P ≤0.001.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis, Recombinant, Expressing, Incubation, Variant Assay, Concentration Assay, Two Tailed Test

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 7. SPR analysis of binding of purified SclWT and Scl single-mutation variants to LRP4. SPR data showing binding of (A) SclWT, (B) SclK75Q, (C) SclK75E, and (D) SclV136D to 3 μg of immobilized LRP4 receptor. Different protein concentrations are represented by different colors.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function

doi: 10.1074/jbc.m110.190330

Figure Lengend Snippet: FIGURE 3. LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 m. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transducedwithlentiviralparticlesharboringshRNAagainstLrp4.ReductionofLrp4RNAlevelswasassessedbyreal-timequantitativePCR,priortotheaddition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

Article Snippet: Recombinant human DKK1 and LRP6 were purchased from R&D Systems.

Techniques: Inhibition, In Vitro, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Activity Assay

Journal: Journal for immunotherapy of cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity.

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: Figure 2 In vitro characterization of anti-GARP antibody PIIO-1. (A) GARP expression on human regulatory T cells and platelets was evaluated by flow cytometry after staining with PIIO-1 at 10 µg/mL. (B) 293 FT cell line was transfected with empty vector (EV), human GARP (hGARP)-expressing vector only or co-transfected with hGARP and latent TGFβ1 expression vectors. GARP expression on indicated cell line was detected by flow cytometry after staining with PIIO-1 at 10 µg/mL. (C) Human GARP sequence was replaced by murine GARP according to the schematic diagram to generate the chimeric constructs of human and murine GARP that were tagged with HA (hemagglutinin) epitope. Transfection efficiency was determined using anti- HA antibody. All constructs were transfected into 293 FT cells. (D) Crystal structure of the GARP (green)-LTGFβ (gray) complex (PDB DOI: 10.2210/pdb6GFF/pdb). The region of PIIO-1 recognition is orange and the residues interacting with LTGFβ are cyan. LTGFβ occludes approximately 30% of the potential antibody binding site and may sterically or allosterically restrict access of the antibody to GARP in the LTGFβ-complexed state. Modeling was carried out using Pymol. (E) Jurkat cell line, made to overexpress hGARP, was incubated with LTGFβ1 along with mIgG1 or PIIO-1 at indicated concentration for 30 min at 37℃. Human LAP expression was detected by flow cytometry. All data are representative of 2–6 independent experiments. GARP, Glycoprotein-A repetitions predominant; LAP, latency-associated peptide.

Article Snippet: Generation of anti-human GARP (hGARP) antibodies The generation of anti- hGARP antibody has been described previously.19 BALB/c mice was immunized with recombinant human GARP (R&D Systems) in Freund’s complete adjuvant and followed by boosting with SP2/0hGARP cells for 2–3 times.

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Sequencing, Construct, Binding Assay, Incubation, Concentration Assay

Journal: Journal for immunotherapy of cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity.

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: Figure 3 PIIO-1 enhanced antitumor efficacy of anti-PD-1 ICB in GARP+ TNBC. (A) Experimental scheme. BALB/c mice were injected with 1×105 4T1-hGARP cells in the mammary fat pad, followed by i.p. injection of 200 µg/mouse of PIIO-1 and/or 150 µg/mouse anti-PD-1 every 3 days. (B) Primary tumor growth curve. (C) Overall survival of mice. (D) Summary of the incidence of tumor-free mice among groups. (E) Lungs were collected and sectioned at experimental end point, then stained with H&E. Representative images from each group of mice are shown. Scale bar, 20 µm. The numbers of visible lung metastatic nodules are quantified. (F) Summary of the incidence of metastasis among groups. (G) Tumors were collected at end point and stained by IHC for pSMAD3, α-SMA. Representative images of tumor tissues from four groups of mice are shown (left). Scale bar, 50 µm. Quantification of the IHC staining is shown (right). (H) Sera from each mouse was collected at end point. Total and active TGFβ level in the sera were assessed by ELISA. (I) Mice with tumor regression following combination therapy were monitored for 300 days, then rechallenged with 5×105 wild-type 4T1 mammary tumor in contralateral mammary fat pad. Naive BALB/c mice without pre-exposure to tumor were used as control. Shown is the overall survival. Tumor curve analysis was performed using repeated measures 2-way analysis of variance. Overall survival is analyzed by log-rank (Mantel-Cox) test. (E, G) were analyzed by paired t-test according to the tumor collection time points. Other data were analyzed by two-tailed Student’s t-test. B, C were corrected for multiple testing using the Tukey procedure. All data are presented as mean±SEM. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

Article Snippet: Generation of anti-human GARP (hGARP) antibodies The generation of anti- hGARP antibody has been described previously.19 BALB/c mice was immunized with recombinant human GARP (R&D Systems) in Freund’s complete adjuvant and followed by boosting with SP2/0hGARP cells for 2–3 times.

Techniques: Injection, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Cell

Article Title: Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis

doi: 10.1016/j.cell.2018.05.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Assay was performed in triplicates in a reaction buffer (50 mM sodium acetate, 8 mM EDTA, 1 mM DTT, 1 μM pefabloc, all Sigma-Aldrich) using 2 μg of protein lysate and 10 μM protease substrate z-LR-AMC (ES008, R&D Systems).

Techniques: In Vivo, Flow Cytometry, Recombinant, Staining, Cell Isolation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Glucose Assay, Reverse Transcription, SYBR Green Assay, Control, Software

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Relationship between LAIR1 expression and intraleukocytic haemozoin. (a) Pairwise comparisons of LAIR1 transcript levels between children with and without pigment-containing monocytes (PCM) and pigment-containing neutrophils (PCN) in the non-SMA and SMA groups were determined using Mann-Whitney U test. Data are presented as box-plots, where the box represents the interquartile range, the line through the box is the median, and whiskers show the 10th and 90th percentiles. LAIR1 transcript levels were lower in children with PCM compared to those without, while LAIR1 transcript levels were comparable between absence/presence of PCN. (b) Temporal kinetics of LAIR1 transcript levels in response to Pf Hz treatment of PBMC from malaria-naïve donors ( n = 6, measured in triplicate). Pairwise comparisons were determined using Student t -test. Significant ( P < .05) differences in transcript levels between no treatment and Pf Hz treatment (10 μg/mL) groups represented by *at 0.5, 2, and 4 h time points. Data represent average of individuals ( n = 3) with each condition performed in triplicate (error bars represent SEM). (c) Immunoblot analysis of non-treated (baseline) and Pf Hz-treated (10 μg/mL) PBMC lysates for 12, 24, and 48 h. (d) Densitometric analysis of normalised cellular LAIR1 protein production presented as mean ± SEM. Across group comparisons analysed using ANOVA. Pairwise comparisons analysed using Student t-test. Data represent average of individuals ( n = 6) with each condition performed in triplicate (error bars represent SEM). LAIR1 protein levels were lower in Pf Hz-treated PBMC lysates compared to no treatment at 12, 24, and 24 h.

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques: Expressing, MANN-WHITNEY, Western Blot

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of intraleukocytic Pf Hz on LAIR1 and SHP-1 phosphorylation. Temporal kinetics of the signalling pathway in response to the different treatment conditions was determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). (a) Human Phospho-immunoreceptor array results showing LAIR1 and SHP-1 phosphorylation upon no treatment (baseline), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. Phosphorylation (spots) in the different conditions with control represented by blue boxes, LAIR1 by red boxes, and SHP-1 by green boxes. (b) Densitometric analysis of human phosphor-immunoreceptor array data. Data presented as (mean ± SEM). Across group comparisons analysed using ANOVA. *indicates significant differences ( P < .05) determined by Student t-test in pLAIR1 and pSHP-1 compared to C1q treatment. Phagocytosis of Pf Hz resulted in a reduction of pLAIR1 relative to C1q (alone) treatment. Similarly, ingestion of Pf Hz also caused a marked decrease in pSHP-1 levels relative to C1q treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques:

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: LAIR1 signalling pathway in malaria. The LAIR1 pathway is activated by attachment of collagen and collagenous ligands (C1q) to LAIR1 extracellular surface. This results in phosphorylation of intracellular LAIR1 ITIM tyrosine residues by Src family kinases. Phosphorylated ITIMs serve as docking sites for recruitment of SHP-1 and SHP-2 phosphatases. SHP-1 and SHP-2 become localized to phosphorylated ITIMs through their regulatory SH2 domains, subsequently inducing their phosphatase activity. Activated SHP-1 has been shown to block activation and nuclear translocation of nuclear factor nuclear factor-kappa beta (NF-κB) through de-phosphorylation of inhibitor of kappa-beta kinase complex (IKK). SHP-1 phosphatase activity also inhibits activation and translocation of IRFs from the cytoplasm to the nucleus by preventing TANK-binding kinase 1 (TANK-1) phosphorylation of interferon regulatory factors (IRFs). These events subsequently block transcription of inflammatory mediator encoding genes with response elements for IRFs and NF-κB. SHP-2 inhibits activation of IRF 8 and blocks expression of phagocyte NADPH oxidase (gp91 PHOX ). LAIR1 inhibitory signalling is regulated by soluble LAIR1 and LAIR2 (soluble homolog of LAIR1) through competition for collagenous ligands. Children with SMA had increased circulating levels of sLAIR1 and sLAIR2 indicative of enhanced receptor shedding. C1q was reduced in children with SMA, thereby, limiting ligand availability. Phagocytosis of Pf Hz antagonizes LAIR1 signalling through down-regulation of LAIR1 ITIM and SHP-1 phosphorylation, but does not alter SHP-2 phosphorylation. Leukocytic ingestion of Pf Hz also decreases LAIR1 transcripts and protein. These events result in NF-κB activation and the consequent production of pro-inflammatory mediators that enhance the pathogenesis of SMA. Red arrows represent ligand-receptor interaction, black solid arrows represent activation, and dashed black arrows represent blockade. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques: Activity Assay, Blocking Assay, Activation Assay, Translocation Assay, De-Phosphorylation Assay, Binding Assay, Expressing

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Leptin receptors in human skeletal muscle.

doi: 10.1152/japplphysiol.01313.2006

Figure Lengend Snippet: Fig. 2. Determination of the expression of human leptin receptor (OB-R) in human skeletal muscle. Protein extracts were prepared from muscle, SAT, and hypothalamus (HIP), and OB-R, perilipin A, and -tubulin protein expression was analyzed by Western blot. A: representative immunoblot assay after incubation with a polyclonal rabbit anti-OB-R antibody specifically raised against the long isoform. B: representative Western blot after incubation with a polyclonal rabbit anti-perilipin A antibody in the same samples used in A. C: representative immunoblot analysis after incubation with the monoclonal mouse anti--tubulin antibody in the same samples used in A. D: densitometric immunosignal values (arbitrary units of band densities) of OB-R bands relative to those obtained for -tubulin.

Article Snippet: The recombinant human (RH) leptin R/Fc chimera, generated from DNA containing the extracellular domain of OB-R (amino acid residues 1-839) fused to the Fc region of human IgG1, was obtained from R&D Systems (McKinley Place).

Techniques: Expressing, Western Blot, Incubation

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Leptin receptors in human skeletal muscle.

doi: 10.1152/japplphysiol.01313.2006

Figure Lengend Snippet: Fig. 3. The anti-OB-R antibody recognized specifically the three OB-R bands detected in the muscle protein extracts. Increasing amounts of recombinant human (RH) leptin R/Fc (RH OB-R) chimera (0, 10, 100, 500 ng) were preincubated with anti-OB-R antibody (1:2,000). OB-R protein expression from muscle extracts was analyzed by immunoblotting using the preincubation solution. A: representative Western blot analysis with different preincubation solutions in the same muscle protein extract (50 g). B: representative immunoblot with the -tubulin antibody as a loading control. C: densitometric percentage of OB-R immnunostaining values (band quenching) in presence of increasing amounts (10, 100, 500 ng) of RH OB-R relative to those observed for a control (0 ng of RH OB-R). *P 0.01 vs. 0 ng of RH OB-R.

Article Snippet: The recombinant human (RH) leptin R/Fc chimera, generated from DNA containing the extracellular domain of OB-R (amino acid residues 1-839) fused to the Fc region of human IgG1, was obtained from R&D Systems (McKinley Place).

Techniques: Recombinant, Expressing, Western Blot, Control