Journal: PLoS ONE

Article Title: Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

doi: 10.1371/journal.pone.0047648

Figure Lengend Snippet: PBMC derived from HLA A2+ patients with CHB were stained with a panel of HLA-A2/HBV multimers and then were stimulated overnight with a pool of HBV peptides of matched specificity to the multimers, followed by intracellular staining for IFN-γor TNF-α. (a) Representative histograms showing levels of Tim-3 (black line) or isotype binding (grey shading) on CD8 T cells binding HLA-A2/HBV peptide multimers or producing IFN-γ upon encounter with HBV peptides. (b) Compiled data from 10 patients with CHB. (c) Tim-3 expression on CD8 T cells binding HLA-A2/HBV peptide multimers or producing TNFa upon stimulation with HBV peptides. (d) FACS plots and (e) summary data showing the induction of caspases (FLICA) and 7AAD in CD8 and CD4 T cells with or without the addition of galectin-9. Active caspases, indicating apoptosis, were determined using a fluorescent-labelled inhibitor of polycaspases (FAM-VAD-FMK, FLICA), and death was identified by 7AAD stain. Early apoptotic events are indicated in the lower right quadrant (FLICA+7AAD−), late apoptotic events in the right upper quadrant (FLICA+7AAD+) and necrotic cells in the left upper quadrant (7AAD+FLICA-). ‘Total death’ was estimated by summing events in these 3 quadrants.

Article Snippet: Immunostaining of cryopreserved sections from HBV infected liver biopsies was performed using a combination of the following primary antibodies Galectin-9 goat polyclonal IgG (R & D Systems) and CD68 mouse monoclonal (KP1, Abcam).

Techniques: Derivative Assay, Staining, Binding Assay, Expressing

Journal: PLoS ONE

Article Title: Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

doi: 10.1371/journal.pone.0047648

Figure Lengend Snippet: Immunohistochemistry of a section from a cryopreserved CHB liver biopsy stained with a polyclonal galectin-9 antibody (shown in brown) at 10X and 40X magnification. (b ) Immunofluorescence of a section from a cryopreserved CHB liver biopsy stained with DAPI (blue, left panel), anti-CD68 mAb (green, central panel) and galectin 9 polyclonal antibody (red, right panel). Double positive staining is indicated in yellow in the lower panel. (c) Kupffer cells were isolated from 4 liver explants and stained with CD14, CD68 and galectin-9 or its isotype. Representative histograms show galectin-9 staining on CD14+CD68+ fraction compared to isotype-matched control. (d) Galectin-9 levels were analysed in the serum of 10 healthy subjects and 42 CHB patients: 16 with ALT<50, 17 with ALT 50–100 and 9 with ALT >100 (mean and SEM shown).

Article Snippet: Immunostaining of cryopreserved sections from HBV infected liver biopsies was performed using a combination of the following primary antibodies Galectin-9 goat polyclonal IgG (R & D Systems) and CD68 mouse monoclonal (KP1, Abcam).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Isolation

Journal: PLoS ONE

Article Title: Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

doi: 10.1371/journal.pone.0147186

Figure Lengend Snippet: (A) The expression of Galctin-9 in HTR8 and K562 cells was determined by RT-PCR. (B) The expression of Galectin-9 in human early pregnant trophoblasts cells was detected by immunohistochemical staining (×400). Formalin fixed paraffin-embedded trophoblasts tissue sections were stained with Goat anti-human Galectin-9 and biotinylated rabbit anti-goat Ig followed by treptavidin-conjugated peroxidase. (C) Knock down Galectin-9 expression by siRNA. The knock down efficacy was determined by RT-PCR. (D) Galectin-9 siRNA significantly increased NK cytotoxicity toward trophoblasts. HTR-8 cells, transfected with NC control or Galectin-9 siRNA, were used as targets and co-cultured with TGF-β1 pretreated pNK cells. The cytotoxicity of NK cells was measured using a Cell Counting Kit-8. (E) HTR-8, transfected with Galectin-9 siRNA, and co-cultured with TGF-β1 pretreated pNK cells at E:T ratio (10:1) with or without rhGalectin-9 (rhGal-9) for 4 hours. The cytotoxicity of NK cells was measured using a Cell Counting Kit-8.

Article Snippet: Goat anti-human TGF-β1, goat anti-human Galectin-9 and Tim-3-Fc fusion protein were purchased from R&D Systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Transfection, Cell Culture, Cell Counting

Journal: PLoS ONE

Article Title: Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

doi: 10.1371/journal.pone.0147186

Figure Lengend Snippet: NK cell alone or co-cultured with HTR-8, HTR-8 treated with Tim-3-Fc or HTR-8 transfected with Galectin-9 siRNA for 3h. Then the co-cultures were harvested and incubated with CD56-FITC. The level of CD107a on CD56 + cells was determined by FACS, one representative result was shown (A). (B) Statistical analysis of CD107a expression (n = 5). *, p < 0.05 compared with no HTR-8 stimulation group, #, p < 0.05 compared with HTR-8 stimulation group.

Article Snippet: Goat anti-human TGF-β1, goat anti-human Galectin-9 and Tim-3-Fc fusion protein were purchased from R&D Systems.

Techniques: Cell Culture, Transfection, Incubation, Expressing

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Free and galectin-9-bound Tim-3 is shed differentially from the cell surface. THP-1 cells were exposed for 16 h to 100 nM PMA; medium was then exchanged for fresh PMA-free medium and cells exposed to the indicated concentrations of GI254023X (ADAM10/17 inhibitor) and BB-94 (matrix metalloproteinase inhibitor). Non treated THP-1 cells were incubated for 16 h after which medium was changed and cells incubated for further 4 h and used as a control. Western blot characterization of galectin-9 and Tim-3 variants (20 kDa fragment (Tim-3 (fr)) and 33 kDa (sTim-3)) was performed in medium collected after final 4 h of incubation of resting and PMA-pre-treated THP-1 cells as outlined in the (A). All the samples were subjected to ELISA-based detection of galectin-9, soluble Tim-3 and Tim-3-galectin-9 complex (B). Images are from one experiment representative of six which gave similar results. Quantitative data represent mean values ± SEM of six independent experiments; * p < 0.05; ** p < 0.01 vs. control.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: PMA activates Tim-3 and galectin-9 production and release as well as generation of Tim-3-galectin-9 complex. THP-1 cells were treated with 100 nM PMA for 16 h. Non-treated THP-1 cells were used as a control. Cells were then harvested and galectin-9 as well as Tim-3 were analyzed in whole cell extracts by Western blot. Both proteins and Tim-3-galectin-9 complexes were analyzed by ELISA in the medium used to treat the cells. The bar diagram on the top shows the comparative analysis (expressed in % control) of galectin-9 and Tim-3-galectin-9 complex levels released by non-treated and PMA-treated THP-1 cells. Images are from one experiment representative of three which gave similar results. Quantitative data are the mean values ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Co-localization of Tim-3 and galectin-9 in PMA-activated THP-1 cells. Co-localization of Tim-3 and galectin-9 was analyzed in non-permeabilized and permeabilized THP-1 cells following 24 h of exposure to 100 nM PMA using confocal microscopy (see for details). Images are from one experiment representative of six which gave similar results.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Confocal Microscopy

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Levels of galectin-9 and soluble Tim-3 are highly increased in blood plasma of AML patients. Galectin-9 and Tim-3 were measured by ELISA in blood plasma obtained from healthy donors and AML patients (A, B, E and F). The levels of Tim-3-galectin-9 complex were measured by ELISA in blood plasma of five randomly picked healthy donors and AML patients (C and G). Tim-3 and galectin-9 were characterized by Western blot in blood plasma from five randomly chosen AML patients (D). Correlations between Tim-3 and galectin-9 as well as between galectin-9 and Tim-3-galectin-9 complex was then determined (H, I, J and K). Images are from one experiment representative of five which gave similar results. Quantitative data represent mean values ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Interaction of Tim-3 with galectin-9 leads to major conformational changes increasing solubility of the protein complex. (A) The schematic structural models of Tim-3 extracellular domain (left) and galectin-9 (right). In the Tim-3 structure, amino acid residues involved in galectin-9-independent binding are highlighted in green. Residues, which are potential targets for glycosylation, are highlighted in red. In galectin-9, sugar molecules, which could potentially bind the protein, located close to the carbohydrate binding sites are shown in green. (B) The SRCD spectroscopy of Tim-3, galectin-9 and Tim-3-galectin-9 interaction (both simulated and real curves are presented). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Solubility, Binding Assay, Spectroscopy

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: LPHN1, PKCα and mTOR pathways are involved in Tim-3 and galectin-9 production and secretion in AML cells. THP-1 cells were exposed to the indicated concentrations of PMA or LTX for 16 h with or without 1 h pre-treatment with the PKCα inhibitor Gö6983 (A, B, D) or the mTOR inhibitor AZD2014 (C, D). Cellular levels of Tim-3 and galectin-9 were analyzed by Western blot. Released Tim-3 and galectin-9 were detected by ELISA. Images are from one experiment representative of three which gave similar results. Quantitative data are the mean values ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. Symbols “a” or “b” are used instead of “*” to indicate differences vs. PMA and LTX-treated cells, respectively.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: FLRT3, a physiological ligand of LPHN1, induces galectin-9 and Tim-3 secretion. (A) THP-1 cells were exposed for 16 h to 10 nM extracellular domain of human recombinant FLRT3 followed by measurement of released Tim-3 and galectin-9 by ELISA. (B) THP-1 cells were exposed to mouse bone marrow (mBM) extracts for 16 h with or without 1 h pre-treatment with 5 μg/ml anti-FLRT3 antibody. The presence of FLRT3 in mBM extracts was confirmed by Western blot analysis. Secreted Tim-3 and galectin-9 were measured by ELISA. (C, left) RCC-FG1 cells express FLRT3 as confirmed by Western blotting. (C, right) RCC-FG1 cells were co-cultured with THP-1 cells at a ratio of 1 THP-1:2 RCC-FG1 with or without 1 h pre-treatment with 5 μg/ml FLRT3 neutralizing antibody. Secreted galectin-9 and Tim-3 were measured by ELISA. Images are from one experiment representative of three which gave similar results. Quantitative data depict mean values ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. Symbols “a” or “b” are used instead of “*” to indicate differences vs. cells treated with mBM extracts or co-cultured with RCC-FG1 cells, respectively.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: LAD2 cells express and externalize Tim-3 and galectin-9. Left panel: surface-based and total Tim-3 and galectin-9 were measured in LAD2 human mast cell sarcoma cells using LI-COR in cell assay (ICA, non-permeabilized cells) and in cell Western (ICW, permeabilized cells). Right panel: protein levels of Tim-3 and galectin-9 were measured in resting and IgE-sensitized LAD2 cells by Western blot. Galectin-9 release was characterized using ELISA. Images are from one experiment representative of three which gave similar results. Quantitative data show mean values ± SEM of three independent experiments; *** p < 0.001 vs. control.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: In-Cell ELISA, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Galectin-9 participates in the formation of an “immunological synapse” between NK cells and LAD2 cells. Primary human NK cells were immobilized on the surface of Maxisorp plates. Cells were then co-incubated for 30 min with LAD2 cells with or without 30 min pre-treatment of LAD2 cells with 5 μg/ml galectin-9 neutralizing antibody (or the same amount of isotype control antibody). LAD2 cells were then visualized using LI-COR assay as outlined in . Images are from one experiment representative of five which gave similar results. Quantitative data represent mean values ± SEM of five independent experiments; * p < 0.05; ** p < 0.01.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Incubation

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Galectin-9 protects myeloid leukemia K562 cells from being killed by primary human NK cells. (A) K562 cells were co-cultured for 16 h with primary human NK cells (at a ratio of 1 K562:2 NK) in the absence or presence of 5 ng/ml galectin-9. Viability of K562 and NK cells was then measured using an MTS test. Images are from one experiment representative of three which gave similar results. Quantitative data represent mean values ± SEM of three independent experiments; *** p < 0.001 vs. control. (B) K562 cells were co-cultured for 16 h with primary human NK cells (at a K562:NK ratio of 1:2) in the presence of different concentrations of galectin-9 (0–5 ng/ml). Cells were imaged using phase-contrast microscopy. The images are from one representative experiment of six ( n = 6), which gave similar results. Scale bar (the same for all images), 50 μm. (C) The NK cell-induced aggregation of K562 cells was quantified as a function of galectin-9 concentration. Left panel: percent of cells found in aggregates in individual cultures and in co-culture. Right panel: the size of cell aggregates in individual cultures and in co-culture. The data represent the mean values ± SD of six independent experiments; *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Cell Culture, Microscopy, Concentration Assay, Co-Culture Assay

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: Cell-derived galectin-9 attenuates AML cell killing activity of primary human NK cells. THP-1 cells were co-incubated with primary human NK cells (ratio – 1 THP-1:2 NK) for 6 h followed by detection of THP-1 cell viability by the MTS test, measurement of activities of granzyme B and caspase 3 in THP-1 cell lysates and released galectin-9 (left panel). Galectin-9 levels from NK cells were determined by Western blot (right panel). Images are from one experiment representative of three which gave similar results. Quantitative data show mean values ± SEM of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001 vs control.

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Derivative Assay, Activity Assay, Incubation, Western Blot

Journal: EBioMedicine

Article Title: The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

doi: 10.1016/j.ebiom.2017.07.018

Figure Lengend Snippet: AML cell-based pathobiochemical pathway showing LPHN1-induced classic activation of PKCα, which triggers translation of Tim-3 and galectin-9 as well as their secretion which is required for immune escape. The interaction of FLRT3 located on the surface of endothelial cells (EC) with LPHN1 leads to the activation of PKCα through the classic Gq/PLC/Ca 2 + pathway. Ligand-bound LPHN1 activates Gq, which in then stimulates PLC. This leads to phosphatidyl-inositol-bisphosphate (PIP2) degradation and production of inositol-trisphospate (IP 3 ) and diacylglycerol (DAG). IP 3 interacts with ER-associated IP 3 receptor (IP3R) leading to Ca 2 + mobilization. PKCα is activated by DAG and Ca 2 + activates mTOR translational pathway through downregulation of TSC1/TSC2. mTOR controls translation of Tim-3 and galectin-9. PKCα also phosphorylates Munc18 exocytosis regulator protein which provokes formation of SNARE complexes that tether vesicles to the plasma membrane. This pre-activates the release machinery, and elevated cytosolic Ca 2 + lead to exocytosis of free and galectin-9-complexed Tim-3. Both types of Tim-3 are differentially shed from the cell surface by proteolytic enzymes. Soluble Tim-3 prevents IL-2 secretion required for activation of NK cells and cytotoxic T cells. Galectin-9 impairs AML cell killing activity of NK cells (and other cytotoxic lymphocytes).

Article Snippet: Single-chain antibody (described above, dilution 1:100) was used to capture the complex and biotinylated goat R&D Systems antibody against galectin-9 (detection antibody) was used to detect galectin-9 bound to Tim-3.

Techniques: Activation Assay, Membrane, Activity Assay

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Predictive value of circulating galectins on the OS and DFS. ( A) Gal-8 and ( B ) gal-9 concentration in the plasma of healthy donors and ovarian cancer patients. ( C ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: galectin negative samples, red bar: galectin positive samples.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Predictive value of circulating galectin-8 and -9 according to CA-125 levels. Kaplan-Meier curves of 5-years OS and 5-years DFS according to the presence of circulating gal-8 and gal-9 in the plasma of ( A ) CA125 LOW or ( B ) CA125 HIGH patients. Blue bar: galectin negative samples, red bar: galectin positive samples.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques:

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Combining gal-8 and gal-9 increases the predictive value for OS and HFS. ( A ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of either or both gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: Gal-8 LOW /Gal-9 LOW , green bar: Gal-8 High /Gal-9 LOW , Yellow bar: Gal-8 LOW /Gal-9 High , Magenta bar: Gal-8 High /Gal-9 HIGH . ( B ) Correlation between the concentration of gal-8 and gal-9 in the plasma of HGSC patients. ( C ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of both gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: Gal-8 LOW /Gal-9 LOW , Gal-8 High /Gal-9 LOW or Gal-8 LOW /Gal-9 High sample; Red bar: Gal-8 High /Gal-9 HIGH samples. Patients with plasma gal-8 ≥ 2.7 ng/ml or plasma gal-9 ≥ 0.73 ng/ml were considered Gal-8 High and Gal-9 High , respectively.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques: Concentration Assay