Journal: Cell Reports Medicine

Article Title: Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors

doi: 10.1016/j.xcrm.2023.101380

Figure Lengend Snippet:

Article Snippet: anti-human LAIR-1-VioBright-FITC (clone REA447) – Lab 1 , Miltenyi Biotec , Cat# 130-106-852; RRID: AB_2657221.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Cell Isolation, Amplification, Multiplexing, Expressing, 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: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) LAIR1 expression in total B cells (left) and the compositions of subsets in LAIR+ and LAIR1-B cells (right) (N=10). (B-D) LAIR1 expression in major B cell subsets (B; N=10), the different class of surface Ig (C; N=5) and major B cell subsets (D; N=9). (E) heatmap and volcano plot generated from RNA-seq data between LAIR1+ and LAIR1- SWM B cells (N=4). Differential gene expression was identified with Log 2 FC >0.5 and adjusted p value <0.05. Upregulated genes in LAIR1- are 278 genes and down-regulated genes in LAIR1- are 218 genes. (F) the potential of PC differentiation induced by TLR7 or TLR9 signaling. (G) the percentages of ANA+ autoreactive B cells in LAIR1+ and LAIR1- memory fraction (N=7). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (F and G) and further adjusted for multiple comparisons using the Benjamini-Hochberg method (B, C and D). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) Phosphorylated LAIR1 induced by its crosslinking with biotinylated anti-LAIR1 antibody and streptavidin (N=5). (B-D) The effects of co-crosslinking with BCR and LAIR1 in naïve B cells (B; N=5), IgM+ USWM B cells (C; N=5) and IgG+ SWM B cells (D; N=5). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests. Asterisks indicate significant differences (* P < 0.05).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques:

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A-B) Representative examples of LAIR1 expression in 7 subsets of B cells (A) and activated naïve B cells and DN2 B cells (B) from HD and SLE patients (each N=9). (C) The positivity of LAIR1 in ANA+ and ANA- B cells. (D) The expression level of LAIR1 in LAIR1 expressing ANA+ autoreactive B cells between HD (N=9) and SLE patients (N=8). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Mann-Whitney U test (A, B and D) or the Wilcoxon signed rank tests (C). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) LAIR1 expression on B cells after 5 days co-culture with cTfh cells activated with anti-CD3/28 Beads in the presence of IL-21R-Fc protein (20 μg/mL) or isotype control (N=5). (B) The effects of IL-21 on LAIR1 expression in naïve B cells co-stimulated with CD40 or CD40/BCR in 3 days culture (N=12). (C) The effects of IL-21 on LAIR1 expression in SWM B cells co-stimulated with CD40 in 5 days culture (N=5). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (C) and further adjusted for multiple comparisons using the Benjamini-Hochberg method (A and B). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, Co-Culture Assay, Control

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) The effects of IL-21 on LAIR1 expression in naïve B cells co-stimulated with TLR9 or TLR9/BCR in 3 days culture (N=7) (B) The effects of IL-21 on LAIR1 expression in ABCs differentiation from naïve B cells in 3 days culture (N=7). (C) The effects of IL-21 on LAIR1 expression in SWM B cells co-stimulated with TLR9 in 5 days culture (N=7). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (B) and further adjusted for multiple comparisons using the Benjamini–Hochberg method (A and C). Asterisks indicate significant differences (* P < 0.05).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) The effects of IL-21 on LAIR1 mRNA expression in naïve B cells co-stimulated with TLR9 or CD40/BCR in 2 days culture (N=6) (B) The effects of IL-21 on LAIR1 mRNA expression in naïve B cells in 2-24 hours culture (N=7). (C) IL-21-induced phosphorylation (left) and nuclear translocation (right) of STAT1/3/5 (N=3). (D) Amplification of IL-21-induced pSTAT3 level by TLR9 or CD40/BCR in the naïve B cell culture (N=4-5). (E-G) the effect of SD36 (1 μM) against STAT family proteins for 3.5 hours (E; N=3), LAIR1 mRNA expression change induced by IL-21 (F; N=6), pSTAT3 and the protein levels of STAT3 (G; N=6) and LAIR1 (H; N=11). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (A, B, D, F and G). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, Phospho-proteomics, Translocation Assay, Amplification, Cell Culture

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) STAT3 putative binding site in LAIR1 promoter region and its sequence (B) the binding of STAT3 of nuclear extract from IL-21 treated naïve B cells to LAIR1 promoter region (sequence #3) (N=3). P values were calculated with the Paired T test (C). Asterisk indicates significant difference (* P < 0.05).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Binding Assay, Sequencing

Journal: Nature

Article Title: Public antibodies to malaria antigens generated by two LAIR1 insertion modalities

doi: 10.1038/nature23670

Figure Lengend Snippet: V gene and insert usage of LAIR1-containing antibodies. Isotype and V(D)J gene usage of heavy chain and light chain of mAbs containing LAIR1 in the switch or in the VDJ region. D genes of the mAbs containing a V(D)J insert cannot always be properly predicted by IMGT. Mutations of the LAIR1 insert are shown as % of identity to genomic unmutated LAIR1 exon. GL = germline; nd = not determined.

Article Snippet: The IEs were then incubated with goat anti-human LAIR1 (R&D Systems, AF2664) for 20 min at room temperature.

Techniques:

Journal: Placenta

Article Title: Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) is reduced with preeclampsia and small for gestational aged fetuses.

doi: 10.1016/j.placenta.2024.08.018

Figure Lengend Snippet: Fig. 1. LAIR1 mRNA and protein levels are reduced in placentas from women with early onset preeclampsia or small for gestational age. (A) LAIR1 mRNA expression was significantly reduced in placentas from women with early onset preeclampsia (blue triangles, n = 78), compared to gestation matched controls (black dots, n = 30). (B) LAIR1 mRNA expression was significantly reduced in placentas from women who delivered an infant small for gestational age (SGA; <10th birthweight centile, red diamonds, n = 32) compared to gestation matched controls (black dots, n = 30). (C) LAIR1 protein expression was significantly reduced in placentas from women with early onset preeclampsia (blue triangles, n = 43), compared to gestation matched controls (black dots, n = 21). (D) LAIR1 protein expression was significantly reduced in placentas from women who delivered an SGA infant (red diamonds, n = 10) compared to gestation matched controls (black dots, n = 21). Data points represent individual patients. Data are presented as box and whisker plot. **P < 0.01, ****P < 0.0001.

Article Snippet: Levels of LAIR1 in plasma and protein lysates were quantified using the Human LAIR1 DuoSet ELISA kit (DY2664–05, R&D System, Minnesota, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Whisker Assay

Journal: Placenta

Article Title: Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) is reduced with preeclampsia and small for gestational aged fetuses.

doi: 10.1016/j.placenta.2024.08.018

Figure Lengend Snippet: Fig. 2. LAIR1 mRNA is reduced in placental explants exposed to hypoxia, but not inflammatory cytokines, IL6 or TNFα. Following IL6 treatment (10 ng/mL), LAIR1 mRNA expression (A) and protein expression in placental lysates (B), and media (C) of placental explants were not altered (red squares) compared to controls (0 ng/mL; black dots). Following TNFα treatment (10 ng/mL), LAIR1 mRNA expression (D) and protein expression in placental lysates (E), and media (F) of placental explants are not altered (blue squares) when compared to controls (black dots, 0 ng/mL). Following exposure to hypoxia (1 % O2), LAIR1 mRNA expression (G) is significantly reduced, while protein expression in placental lysates (H), and media (I) of placental explants were not significantly altered (green squares) compared to controls (black dots, 8 % O2). All experiments were done in triplicate, n = 5 patients. Data are presented as mean ± SEM. **P < 0.01.

Article Snippet: Levels of LAIR1 in plasma and protein lysates were quantified using the Human LAIR1 DuoSet ELISA kit (DY2664–05, R&D System, Minnesota, USA) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Effects of shRNA-mediated silencing of expression of indicated ITIM receptors on growth of MV4-11 and NB4 AML cells. The cells were counted on day 6 post-infection, and normalized to cells treated with scrambled shRNAs (mean ± s.e.m., Student's t-test; n=3 samples, sh-LAIR1 *** p < 0.0001; sh-KIR3DL1 ** p = 0.0051; sh-PECAM1 ** p = 0.0061; sh-SIGLEC11 ** p = 0.0089; sh-SIGLEC6 * p = 0.0326; sh-LILRB1 * p = 0.0412; sh-LILRB2 ** p = 00059; sh-LILRB3 *** p < 0.0001; sh-LILRB4 *** p < 0.0001; sh-KIR2DL2 * p = 0.0182; sh-KIRG1 ** p = 0.0096). B, Flow cytometry analysis showing that LAIR1 is highly expressed on the cell surfaces of RCH-ACV, 697, Kasumi2, MV4-11, U937, and THP1 human leukemia cells but not on K562 cells. X-axis indicates LAIR1 expression, and Y-axis indicates cell numbers. The bar graph summarized MFIs of cell surface LAIR1 (relative fold changes to isotype control). C, Endogenous LAIR1 expression was inhibited by lentivirus vector-based expression of different shRNAs (226, 277, 593) in MV4-11 cells as determined by western blotting at 48 hours after lentiviral infection. D, Cell-surface LAIR1 expression was inhibited by shRNAs in MV4-11 cells as determined by flow cytometry at 48 hours after lentiviral infection. MFIs (obtained after subtraction of the background MFI of the unstained control) indicate LAIR1 expression levels; GFP expression indicates infection. Data are from a single experiment, representative of 3 independent experiments. E, Treatment with shRNA targeting lair1 inhibited the growth of MV4-11 cells. F, Expression from Flag- lair1 -6m is not silenced by shRNA 226 as determined by western blotting. G, Rescue of lair1 -knockdown phenotype. RFP-tagged mutant lair1 (6m) infected MV4-11 cells are resistant to the shRNA 226-induced growth inhibition. H, Apoptosis is increased in MV4-11 cells after treatment with shRNA 226 to inhibit lair1 expression (mean ± s.e.m., Student's t-test; n=3 samples, Day 2 * p = 0.0112; Day 4 *** p < 0.0001; Day 6 *** p < 0.0001; Day 9 *** p < 0.0001). I, Treatment with each shRNA targeting lair1 inhibited the growth of 697 cells. J, Lair1 knockdown inhibited the growth of THP1, U937, RCH-ACV, and Kasumi2 cells as measured on day 6. In Figs 1B, E, G, I, and J, data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: shRNA, Expressing, Infection, Flow Cytometry, Control, Plasmid Preparation, Western Blot, Knockdown, Mutagenesis, Inhibition

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, MV4-11 cells (1×10 6 cells) were infected with virus designed to express GFP and either scrambled shRNA or shRNA 226. GFP + cells were collected and transplanted into NSG mice (n = 7 mice) one day post-infection. Left panels show representative flow cytometry plots indicating decreased BM engraftment of MV4-11 cells treated with shRNA targeting lair1 . Staining with anti-human CD45 and anti-LAIR1 antibodies confirmed engraftment was from transplanted human leukemia cells. The percentages of each population are indicated in red numbers, and the median fluorescent intensity of LAIR1 expression is indicated in black numbers. The panel on the right plots percentages of GFP + cells in BM, spleen, liver, and PB at 1 month after transplantation (mean ± s.e.m., Student's t-test; n=7 samples, BM *** p < 0.0001; Spleen ** p = 0.0004; Liver * p = 0.0233; PB * p = 0.01471). B, Comparison of the sizes of spleens of the mice transplanted with control MV4-11 leukemia cells or cells expressing shRNA targeting lair1 (mean ± s.e.m., Student's t-test; n=7 samples, ** p = 0.0028). C, GFP + 697 cells infected by scrambled shRNA or shRNA 226 virus were collected, and 5×10 5 cells were transplanted into NSG mice (n = 5 mice for Scramble, n = 7 mice for lair1 null). Left are representative flow cytometry plots showing the decreased BM engraftment of lair1- knockdown 697 cells. Human CD45 and LAIR1 antibody staining confirmed engraftment was from transplanted human leukemia cells. The percentages of each population are indicated in red numbers, and the median fluorescent intensity of LAIR1 expression is indicated in black numbers. Shown on the right are percentages of GFP + cells in BM, spleen, liver, and PB at 1 month after transplantation (mean ± s.e.m., Student's t-test; n = 5 mice for Scramble, n = 7 mice for lair1 null, BM ** p = 0.0004; Spleen ** p = 0.0002; Liver *** p <0.0001; PB * p = 0.0057). D, Comparison of the sizes of livers of the mice transplanted with control or lair1 -knockdown 697 leukemia cells (mean ± s.e.m., Student's t-test; n = 5 mice for Scramble, n = 7 mice for lair1 null, * p = 0.0048). E, Summary of the effects of lair1 silencing in the indicated human leukemia cell lines on inhibition of cell growth in vitro and xenograftment in NSG mice.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Infection, Virus, shRNA, Flow Cytometry, Staining, Expressing, Transplantation Assay, Comparison, Control, Knockdown, Inhibition, In Vitro

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Survival curves of mice receiving 3,000 pooled YFP + BM cells that were collected from primary recipients transplanted with WT or lair1 -null MLL-AF9 AML cells (n = 10 mice; p = 0.00002, log-rank test). B, Comparison of the spleen sizes of mice that were transplanted with WT or lair1 -null MLL-AF9 AML cells collected from secondary recipients at 18 days or 28 days after transplantation (mean ± s.e.m., Student's t-test; n = 5 mice, 18 days * p = 0.0245; 28 days ** p = 0.0006). C, Summary of the percentages of YFP + WT and lair1 -null MLL-AF9 leukemia cells in PB (open squares or open diamonds) or BM (red squares or red diamonds) of secondarily transplanted mice over time. Mice that were moribund and euthanized are indicated by red spots, in which the percentages of YFP + cells in BM (instead of PB) were used in this analysis (n = 10 mice). D, Summary of percentages of YFP + AML cells in PB, BM, and spleen of secondary recipient mice transplanted with the WT or lair1 -null MLL-AF9 AML cells at day 18 (upper panel) and at day 28 (lower panel) post-transplant (mean ± s.e.m., Student's t-test; n = 5 mice, 18 days PB * p = 0.0124; BM * p = 0.0243; Spleen * p = 0.0332; 28 days PB ** p = 0.00035; BM *** p < 0.0001; Spleen ** p =0.0014). E, Histological analysis of AML infiltration in the livers, spleens, and PB of mice after secondary transplant of WT and lair1 -null AML cells at 4 weeks and 20 weeks (hematoxylin/eosin staining for livers and spleens, HEMA 3 staining for cytospin-prepared PB samples). Shown are representative images from at least three similar images. Scale bar is 100 μM. F, Survival curves of mice transplanted with 1×10 5 pooled GFP + BM cells that were collected from primary recipients transplanted with WT or lair1 -null AML1-ETO9a cells (n = 10 mice; p = 0.00012, log-rank test). G, Percentages of AML1-ETO9a GFP + leukemia cells in BM at 1 month after secondary transplantation (mean ± s.e.m., Student's t-test; n = 10 mice; *** p < 0.0001).

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Comparison, Transplantation Assay, Staining

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Representative flow cytometry plots showing that secondarily transplanted mice receiving lair1 -null AML cells had markedly decreased percentages of Mac1 + Kit + cells and increased differentiated B220 + and CD3 + cells in YFP + compartments compared to mice receiving WT AML cells. Three pairs of mice were used for the comparison. B-D, Percentages of YFP + Mac-1 + Kit + , YFP + Mac-1 + Gr-1 + , YFP + B220 + , and YFP + CD3 + cells in (B) BM, (C) PB, and (D) spleen of secondary recipient mice transplanted with the WT or lair1 -null MLL-AF9 AML cells at day 18 post-transplant (mean ± s.e.m., Student's t-test; n = 5 mice; (B) Mac1+/Gr1+ * p = 0.0195; Mac1+/kit+ ***p <0.0001; B220+ ** p = 0.0025; CD3+ * p = 0.0256; (C) Mac1+/Gr1+ * p = 0.0211; Mac1+/kit+ ***p <0.0001; CD3+ ***p <0.0001; (D) Mac1+/Gr1+ * p = 0.0342; Mac1+/kit+ **p =0.0015; B220+ *** p <0.0001; CD3+ ** p = 0.0027). E, Comparison of colony-forming activity of WT and lair1 -null MLL-AF9 + BM cells during serial replating. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. F, Left panel shows typical morphology of WT and lair1 -null colonies formed by YFP + Mac-1 + Kit + cells collected from secondarily transplanted mice at 28 days post-transplant. The right panel is a comparison of colony-forming activity of Mac1 + Kit + cells from MLL-AF9 + bone marrow cells harvested at 28 days after secondary transplantation. Scale bar is 10 μM. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. G, Survival curve of tertiary recipient mice transplanted with 1,000 YFP + Mac1 + Kit + cells from MLL-AF9 + WT or lair1 -null BM cells harvested at 28 days after secondary transplantation (n = 10 mice; p = 0.000022, log-rank test). H, Flow cytometry analysis of apoptosis in mouse MLL-AF9 YFP + (AML) cells and YFP - (normal) cells. Early apoptosis was detected as Annexin V positive/PE positive/7-AAD negative staining and late apoptosis was detected as Annexin V positive/PE positive/7-AAD positive staining (mean ± s.e.m., Student's t-test; n =3 samples, *** p < 0.0001). I, Limiting dilution assays comparing the frequencies of AML stem cells in WT and lair1 -null MLL-AF9 + AML. The indicated YFP + WT and lair1 -null MLL-AF9 + BM cells that were collected from primary recipients were co-transplanted with 2×10 5 bone marrow competitor cells into lethally irradiated recipients. The CRUs were calculated by L-Calc software.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Flow Cytometry, Comparison, Activity Assay, Transplantation Assay, Negative Staining, Staining, Irradiation, Software

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Phospho-SHP-1 and total SHP-1 levels in lair1 -null and control MLL-AF9 + BM cells from both primarily and secondarily transplanted mice were determined by western blotting. The experiment was repeated for 5 times, which gave similar results. B, Retrovirally-expressed SHP-1 increased CFU numbers of lair1 -null AML cells compared to cells that were infected with control or SHP-2-expressing viruses in colony-forming assays. Retrovirally-expressed SHP-1 and SHP-2 had similar levels as endogenous proteins in WT controls. C-D, Retrovirally-expressed SHP-1 rescued lair1 -null phenotype upon tertiary transplantation. MLL-AF9 + WT or lair1 -null BM cells in secondarily transplanted mice were harvested at 28 days and were infected by SHP-1 encoding or control virus. (C) Survival curves of mice transplanted with 3,000 of these ectopically SHP-1-expressing, SHP-2-expressing, or control cells (n= 5 mice; p < 0.0001, log-rank test). (D) Percentages of retrovirus-infected (GFP + ) AML cells in PB of tertiary recipient mice after 28 days of transplantation (mean ± s.e.m., Student's t-test; n =3 samples, *** p < 0.0001). E-G, Bone marrow cells isolated from 5-fluorouracil pretreated SHP-1 fl/fl mice were transformed with retroviral MLL-AF9 and then transduced by infection with empty vector or Cre-expressing vector to generate control (SHP-1 fl/fl + Con) or SHP-1-deficient (SHP-1 fl/fl + Cre) MLL-AF9 AML cells. (E) Survival curves of mice receiving 3,000 GFP + SHP-1 fl/fl + Con or SHP-1 fl/fl + Cre MLL-AF9 AML cells (n = 10; p < 0.0001, log-rank test). (F) Comparison of percentages of GFP + AML cells in the PB from SHP-1 fl/fl + Con and SHP-1 fl/fl + Cre MLL-AF9 AML cells injected mice (mean ± s.e.m., Student's t-test; n = 10 mice; ** p = 0.0067). (G) Comparison of colony-forming abilities of SHP-1 fl/fl + Con and SHP-1 fl/fl + Cre MLL-AF9 AML cells. H, Bone marrow cells isolated from 5-fluorouracil pretreated SHP-1 fl/fl mice were transduced by infection with empty vector or Cre expression vector to generate control (SHP-1 fl/fl + Con) or SHP-1-deficient (SHP-1 fl/fl + Cre) cells without the MLL-AF9 transformation. Shown is the comparison of colony-forming abilities between control and SHP-1-deficient bone marrow cells. I, Total SHP-1 and CAMK1 levels in WT and lair1 -null BM cells were determined by western blotting. In Fig. 5B, G, and H, data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Control, Western Blot, Infection, Expressing, Transplantation Assay, Virus, Isolation, Transformation Assay, Retroviral, Plasmid Preparation, Comparison, Injection

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Retrovirally-expressed WT SHP-1 or mutants C 453 S or 4YF (278, 303, 538, 566), but not the SHP-1 PTP domain (PTPc), increased CFU numbers of lair1 -null AML cells. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. B-C, Retrovirally-expressed SHP-1 WT, C 453 S, and 4YF, but not SHP-1 PTPc, rescued lair1 -null phenotype upon transplantation. The green, blue, and purple dotted lines representing SHP-1 WT, C 453 S, and 4YF virus infected leukemia BM cells reversed lair1 -null MLL-AF9 + leukemia development. The red dotted line representing control virus infected leukemia BM cells and black dotted line representing SHP-1 PTP domain virus infected leukemia BM cells were unable to rescue (n= 5 mice; (B) p < 0.0001, log-rank test; (C) *** p < 0.0001, mean ± s.e.m., Student's t-test). D, Total CAMK1 and CAMK5 levels in lair1 -null and control MLL-AF9 + BM cells from both primarily and secondarily transplanted mice were determined by western blotting. E, Retrovirally-expressed CAMK1 increased CFU numbers of lair1 -null AML cells. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. F-G, Retrovirally-expressed CAMK1 rescued lair1 -null phenotype upon transplantation. MLL-AF9 + WT or lair1 -null BM cells in secondarily transplanted mice were harvested at 28 days and were infected by CAMK1-encoding or control virus (n= 5 mice, (F) p < 0.0001, log-rank test; (G) *** p < 0.0001, mean ± s.e.m., Student's t-test). H, Endogenous CAMK1 and SHP-1 interact with each other as determined by bi-directional pull down assays. WT MLL-AF9 + BM cells (1×10 7 cells) were lysed by RIPA buffer, and indicated antibodies were used for immunoprecipition and western blot. I, The C-terminal phosphatase-active domain of SHP-1 did not bind to CAMK1 in transfected 293T cells. The indicated Flag-tagged WT or mutant SHP-1 proteins were overexpressed in 293 cells. Flag antibody was used to precipitate SHP-1 proteins, and the Flag or CAMK1 antibodies were used in western blots.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Transplantation Assay, Virus, Infection, Control, Western Blot, Transfection, Mutagenesis

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Phospho-CREB and total CREB levels in lair1 -null and control MLL-AF9 + BM cells from both primarily and secondarily transplanted mice were determined by western blotting. B, Retrovirally-expressed CREB WT, but not CREB S129A, S133A, or S129/S133A, increased CFU numbers of lair1 -null AML cells. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results. C-D, Retrovirally-expressed CREB WT, but not CREB mutants, rescued lair1 -null phenotype upon transplantation. While the red dotted line representing CREB WT virus infected leukemia BM cells reversed the lair1 -null MLL-AF9 + leukemia development, the black, blue, brown, and green dotted lines representing control, CREB S129A, CREB S133A, CREB S129/133A virus each infected leukemia BM cells were unable to do so (n= 5 mice; (C) p < 0.0001, log-rank test; (D) *** p < 0.0001, mean ± s.e.m., Student's t- test). E, Treatment with a CREB inhibitor XX15 reduced CREB phosphorylation and the colony forming ability of WT AML cells. The molecule structure of the CREB inhibitor XX15 is shown. Data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Control, Western Blot, Transplantation Assay, Virus, Infection, Phospho-proteomics

Journal: Nature cell biology

Article Title: The ITIM-containing receptor LAIR1 is essential for acute myeloid leukemia development

doi: 10.1038/ncb3158

Figure Lengend Snippet: A, Expression of lair1 mRNA differs significantly in BM and PB of AML patients (n = 7 BM or 19 PB respectively) from those in normal samples (n = 10 BM or 9 PB; mean ± s.e.m., Student's t-test; BM * p = 0.0329; PB * p = 0.0421). B, Representative flow cytometry plots showing different LAIR1 expression patterns in normal mononuclear cells (Cord blood) and primary AML cells (type 1 and type 2). A representative plot of a flow cytometry analysis (from at least 3 similar images) of an type 1 sample shows that cells are present that express both high and low levels of LAIR1. Type 2 samples mainly contain LAIR1 high cells. Data on these primary AML samples are listed in sTable 3. C, Summary of percentages of hCD45 + cells in PB of NSG mice transplanted with LAIR1 low or LAIR1 high primary human AML cells from 2 patient samples at 4 months after transplantation (mean ± s.e.m., Student's t-test, n= 5 mice, *** p < 0.0001). D&G, Expression of shp-1 or camk1 mRNA negatively correlates with the overall survival of AML patients. n = 82 samples for high and n = 83 samples for low (TCGA database); (D) p =0.0205; (G) p =0.0037, log-rank test). E&H, Treatment with shRNAs targeting shp-1 or camk1 inhibited the growth of MV4-11 cells. F&I, Increased apoptosis in shp-1 -silenced (by shRNA 698) or camk1 -silenced MV4-11 cells (mean ± s.e.m., Student's t-test; n =3 samples, (F) Day 3 *p =0.0324; Day 9 ** p = 0.0019; (I) Day 3 *p =0.0302; Day 9 ** p = 0.0031). J, Endogenous SHP-1 and CAMK1 expression was silenced with shRNAs (226, 277) in MV4-11 cells as determined by western blotting at 72 hours after shRNA-encoding lentiviral infection. K, Expression of lair1 , camk1 , and shp-1 in 165 AML patients (TCGA database) showed highly significant positive correlations. These correlations were also supported by clustering analysis. L, CREB inhibitor XX15 treatment inhibited the growth of MV4-11 cells. M, Flow cytometry analysis of apoptosis of MV4-11, U937, and 697 cells after one day of treatment with XX15 (mean ± s.e.m., Student's t-test; n =3 samples, MV4-11 *p =0.0411; U937 ** p = 0.0216; 697 *p =0.0294). In Fig. 8E, H, and L, data from one experiment with n=3 technical replicate samples are shown. The experiment was repeated 3 times with similar results.

Article Snippet: For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol , and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.

Techniques: Expressing, Flow Cytometry, Transplantation Assay, shRNA, Western Blot, Infection

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: medRxiv

Article Title: Polymerized type I collagen down-regulates STAT-1 phosphorylation through engagement to LAIR-1 in M1-macrophages avoiding long COVID

doi: 10.1101/2023.07.01.23292108

Figure Lengend Snippet: Binding of PTIC to LAIR-1 and its effect on M1-macrophages. (A) Binding assay of PTIC and native porcine type I collagen (CI) to LAIR-1. M1-macrophages treated with (B) anti-LAIR-1 antibody (1:100) for 24 hs or (D) anti-LAIR-1 antibody (1:100) and 10% PTIC for 24 h. Expression of CD14, CD16, CD163, IL-10 in cultures of M1-macrophage treated with (C) anti-LAIR-1 antibody or (E) anti-LAIR-1 antibody and 10% PTIC for 24 h. (F) M1 (CD16 + /CD36 + /CD86 + /IL-1β + ) and M2 (CD14 + /CD16 hi /CD163 + /IL-10 + ) cell percentage.

Article Snippet: Binding assays were performed by incubating various concentrations of recombinant human LAIR-1 (R&D #2664-LR-050) overnight at 4°C in 96 micro-wells plates coated with 5 μg/ml native porcine type I collagen or PTIC and blocked with 5% fat-free milk-PBS.

Techniques: Binding Assay, Expressing

Journal: medRxiv

Article Title: Polymerized type I collagen down-regulates STAT-1 phosphorylation through engagement to LAIR-1 in M1-macrophages avoiding long COVID

doi: 10.1101/2023.07.01.23292108

Figure Lengend Snippet: Binding of PTIC to LAIR-1 induces downregulation of STAT-1 phosphorylation. (A) Western blotting relative expression of STAT-1 and p-STAT1. (B) Relative expression of STAT-1 and pSTAT-1. Both were normalized for actin and were adjusted to 1 unit. (C) Effect of STAT-1 phosphorylation by activating LAIR-1 with anti-LAIR-1 antibody in M1-macrophages. (D) Relative expression of phosphorylated STAT-1. Results were normalized for actin and were adjusted to 1 unit.

Article Snippet: Binding assays were performed by incubating various concentrations of recombinant human LAIR-1 (R&D #2664-LR-050) overnight at 4°C in 96 micro-wells plates coated with 5 μg/ml native porcine type I collagen or PTIC and blocked with 5% fat-free milk-PBS.

Techniques: Binding Assay, Western Blot, Expressing