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Novus Biologicals rat anti emmprin
Rat Anti Emmprin, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rat anti emmprin - by Bioz Stars, 2026-06

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Figure 1. EMMPRIN is highly expressed in rat serum and vitreous humor samples. Rats in DR + SP-8356 or DR + saline group were intraperitoneally injected with 65 mg/kg streptozotocin to induce diabetes and then administered with 50 mg/kg SP-8356 (an inhibitor for EMMPRIN) or saline. Rats in sham + SP-8356 or sham + saline group received injection of 65 mg/kg saline followed by administration of 50 mg/kg SP-8356 or saline. (a) ELISA was performed to measure the content of EMMPRIN in rat serum samples of each group. (b) The protein level of EMMPRIN in rat serum samples was measured by western blotting. (c) Relative protein level of EMMPRIN in rat serum was calculated with normalization to β-actin. (d) EMMPRIN level in rat vitreous samples was measured using ELISA. (e) Western blotting was conducted to measure EMMPRIN protein level in rat vitreous samples of each group. (f) Relative protein level of EMMPRIN in vitreous humor was normalized to β-actin level. Signiﬁcance among multiple groups was evaluated by one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± standard deviation. ***p < 0.001 versus sham + saline group, ###p < 0.001 versus DR + saline group. DR: Diabetic retinopathy. EMMPRIN: extracellular matrix metalloproteinase inducer. ELISA: Enzyme- linked immunosorbent assay.

Journal: Diabetes & vascular disease research

Article Title: EMMPRIN aggravates angiogenesis and blood-retina barrier injury by regulating matrix metalloproteinases in diabetic retinopathy.

doi: 10.1177/14791641251324556

Figure Lengend Snippet: Figure 1. EMMPRIN is highly expressed in rat serum and vitreous humor samples. Rats in DR + SP-8356 or DR + saline group were intraperitoneally injected with 65 mg/kg streptozotocin to induce diabetes and then administered with 50 mg/kg SP-8356 (an inhibitor for EMMPRIN) or saline. Rats in sham + SP-8356 or sham + saline group received injection of 65 mg/kg saline followed by administration of 50 mg/kg SP-8356 or saline. (a) ELISA was performed to measure the content of EMMPRIN in rat serum samples of each group. (b) The protein level of EMMPRIN in rat serum samples was measured by western blotting. (c) Relative protein level of EMMPRIN in rat serum was calculated with normalization to β-actin. (d) EMMPRIN level in rat vitreous samples was measured using ELISA. (e) Western blotting was conducted to measure EMMPRIN protein level in rat vitreous samples of each group. (f) Relative protein level of EMMPRIN in vitreous humor was normalized to β-actin level. Signiﬁcance among multiple groups was evaluated by one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± standard deviation. ***p < 0.001 versus sham + saline group, ###p < 0.001 versus DR + saline group. DR: Diabetic retinopathy. EMMPRIN: extracellular matrix metalloproteinase inducer. ELISA: Enzyme- linked immunosorbent assay.

Article Snippet: To determine the concentration of EMMPRIN, TNFα, IL-6 and IL-1β in rat vitreous samples or serum samples, rat EMMPRIN ELISA kit (orb780849, biorbyt, Cambridge, UK), rat TNFα ELISA kit (ab236712, Abcam), rat IL-6 ELISA kit (ab234570, Abcam), and rat IL-1β ELISA kit (ab255730, Abcam) were used.

Techniques: Saline, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Figure 2. The silencing of EMMPRIN diminishes proinﬂammatory cytokine levels. (a-c) ELISA was performed to measure concentrations of inﬂammatory cytokines, including TNF-α (a), IL-6 (b) and IL-1β (b), in rat vitreous samples. (d–e) RT-qPCR was conducted to assess mRNA levels of inﬂammatory factors (TNF-α, IL-6, and IL-1β) in rat vitreous humor. Signiﬁcance among multiple groups was evaluated using one-way analysis of variance followed by Tukey’s post hoc test. Data are displayed as the mean ± standard deviation. ***p < 0.001 versus sham + saline group; ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. ELISA: Enzyme-linked immunosorbent assay. TNF-α: Tumor Necrosis Factor alpha. IL-6: Interleukin-6. IL-1β: Interleukin-1 beta. RT-qPCR: reverse transcription quantitative polymerase chain reaction. DR: Diabetic retinopathy.

Journal: Diabetes & vascular disease research

Article Title: EMMPRIN aggravates angiogenesis and blood-retina barrier injury by regulating matrix metalloproteinases in diabetic retinopathy.

doi: 10.1177/14791641251324556

Figure Lengend Snippet: Figure 2. The silencing of EMMPRIN diminishes proinﬂammatory cytokine levels. (a-c) ELISA was performed to measure concentrations of inﬂammatory cytokines, including TNF-α (a), IL-6 (b) and IL-1β (b), in rat vitreous samples. (d–e) RT-qPCR was conducted to assess mRNA levels of inﬂammatory factors (TNF-α, IL-6, and IL-1β) in rat vitreous humor. Signiﬁcance among multiple groups was evaluated using one-way analysis of variance followed by Tukey’s post hoc test. Data are displayed as the mean ± standard deviation. ***p < 0.001 versus sham + saline group; ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. ELISA: Enzyme-linked immunosorbent assay. TNF-α: Tumor Necrosis Factor alpha. IL-6: Interleukin-6. IL-1β: Interleukin-1 beta. RT-qPCR: reverse transcription quantitative polymerase chain reaction. DR: Diabetic retinopathy.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Standard Deviation, Saline, Reverse Transcription, Real-time Polymerase Chain Reaction

Figure 3. Inhibition of EMMPRIN attenuates the overproduction of MMPs. (a) Western blotting was performed to quantify protein levels of MMP1, MMP2, and MMP9 in rat vitreous samples. (b–d) Relative MMP1 (b), MMP2 (c), and MMP9 (d) protein levels were quantitated and normalized to β-actin. (e–g) RT-qPCR was performed to assess mRNA levels of MMP1, MMP2, and MMP9 in rat vitreous humor. Comparisons among multiple groups was evaluated by one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± standard deviation. ***p < 0.001 versus sham + saline group, ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. MMPs: matrix metalloproteinases. RT-qPCR: reverse transcription quantitative polymerase chain reaction. mRNA: messenger RNA. DR: Diabetic retinopathy.

Journal: Diabetes & vascular disease research

Article Title: EMMPRIN aggravates angiogenesis and blood-retina barrier injury by regulating matrix metalloproteinases in diabetic retinopathy.

doi: 10.1177/14791641251324556

Figure Lengend Snippet: Figure 3. Inhibition of EMMPRIN attenuates the overproduction of MMPs. (a) Western blotting was performed to quantify protein levels of MMP1, MMP2, and MMP9 in rat vitreous samples. (b–d) Relative MMP1 (b), MMP2 (c), and MMP9 (d) protein levels were quantitated and normalized to β-actin. (e–g) RT-qPCR was performed to assess mRNA levels of MMP1, MMP2, and MMP9 in rat vitreous humor. Comparisons among multiple groups was evaluated by one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± standard deviation. ***p < 0.001 versus sham + saline group, ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. MMPs: matrix metalloproteinases. RT-qPCR: reverse transcription quantitative polymerase chain reaction. mRNA: messenger RNA. DR: Diabetic retinopathy.

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Standard Deviation, Saline, Reverse Transcription, Real-time Polymerase Chain Reaction

Figure 4. EMMPRIN depletion increases levels of tight junction proteins. (a) Levels of tight junction proteins (ZO-1, Occludin, and Claudin-1) were identiﬁed by western blotting. (b–d) ZO-1 (b), Occludin (c), and Claudin-1 (d) levels were quantiﬁed by ImageJ software with normalization to β-actin. (E-G) The mRNA levels of tight junction proteins (ZO-1, Occludin, and Claudin-1) were assessed using RT-qPCR. Signiﬁcance among multiple groups was analyzed by one-way analysis of variance followed by Tukey’s post hoc test. Data are displayed as the mean ± standard deviation. ***p < 0.001 versus sham + saline group; ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. ZO-1: zonula occludens-1. RT-qPCR: reverse transcription quantitative polymerase chain reaction. mRNA: messenger RNA. DR: Diabetic retinopathy.

Journal: Diabetes & vascular disease research

Article Title: EMMPRIN aggravates angiogenesis and blood-retina barrier injury by regulating matrix metalloproteinases in diabetic retinopathy.

doi: 10.1177/14791641251324556

Figure Lengend Snippet: Figure 4. EMMPRIN depletion increases levels of tight junction proteins. (a) Levels of tight junction proteins (ZO-1, Occludin, and Claudin-1) were identiﬁed by western blotting. (b–d) ZO-1 (b), Occludin (c), and Claudin-1 (d) levels were quantiﬁed by ImageJ software with normalization to β-actin. (E-G) The mRNA levels of tight junction proteins (ZO-1, Occludin, and Claudin-1) were assessed using RT-qPCR. Signiﬁcance among multiple groups was analyzed by one-way analysis of variance followed by Tukey’s post hoc test. Data are displayed as the mean ± standard deviation. ***p < 0.001 versus sham + saline group; ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. ZO-1: zonula occludens-1. RT-qPCR: reverse transcription quantitative polymerase chain reaction. mRNA: messenger RNA. DR: Diabetic retinopathy.

Techniques: Western Blot, Software, Quantitative RT-PCR, Standard Deviation, Saline, Reverse Transcription, Real-time Polymerase Chain Reaction

Figure 5. EMMPRIN knockdown suppresses protein levels of angiogenic factors. (a) Protein levels of angiogenic factors, including VEGF, FGF2, and TGFβ1, in rat vitreous humor were quantiﬁed using western blotting. (b–d) Relative VEGF, FGF2, and TGFβ1 protein levels in each group were calculated and normalized to β-actin level. (e–g) RT-qPCR was performed to measure mRNA expression of angiogenic factors (VEGF, FGF2, and TGFβ1) in rat vitreous humor. Signiﬁcance among multiple groups was evaluated by one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± standard deviation. ***p < 0.001 versus sham + saline group; ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. VEGF: vascular endothelial growth factor. FGF2: Fibroblast growth factor 2. TGFβ1: Transforming growth factor beta 1. RT-qPCR: reverse transcription quantitative polymerase chain reaction. mRNA: messenger RNA. DR: Diabetic retinopathy.

Journal: Diabetes & vascular disease research

Article Title: EMMPRIN aggravates angiogenesis and blood-retina barrier injury by regulating matrix metalloproteinases in diabetic retinopathy.

doi: 10.1177/14791641251324556

Figure Lengend Snippet: Figure 5. EMMPRIN knockdown suppresses protein levels of angiogenic factors. (a) Protein levels of angiogenic factors, including VEGF, FGF2, and TGFβ1, in rat vitreous humor were quantiﬁed using western blotting. (b–d) Relative VEGF, FGF2, and TGFβ1 protein levels in each group were calculated and normalized to β-actin level. (e–g) RT-qPCR was performed to measure mRNA expression of angiogenic factors (VEGF, FGF2, and TGFβ1) in rat vitreous humor. Signiﬁcance among multiple groups was evaluated by one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± standard deviation. ***p < 0.001 versus sham + saline group; ###p < 0.001 versus DR + saline group. EMMPRIN: extracellular matrix metalloproteinase inducer. VEGF: vascular endothelial growth factor. FGF2: Fibroblast growth factor 2. TGFβ1: Transforming growth factor beta 1. RT-qPCR: reverse transcription quantitative polymerase chain reaction. mRNA: messenger RNA. DR: Diabetic retinopathy.

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Expressing, Standard Deviation, Saline, Reverse Transcription, Real-time Polymerase Chain Reaction

(A) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT , SOD1 G93A , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human SOD1 WT or SOD1 G93A and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=5 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT , SOD1 G93A , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human SOD1 WT or SOD1 G93A and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=5 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

(A-D) Representative western blot of (A) lysates from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of PPIA (B) , low-glycosylated (37kDa) (C) and high-glycosylated (50kDa) (D) forms of EMMPRIN (EMN). Data are mean±SEM of n=3 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (E-G) Representative western blot of (E) media from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of extracellular PPIA (ePPIA) (F) and soluble EMMPRIN (sEMN) (G) . Data are mean±SEM of n=3-4 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (H) Luciferase assay for NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 72h and treated with 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3 independent experiments expressed as fold of Mock untreated cells. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: Target protein intensity was normalized on total transferred proteins (TTP). Relative luminescence units (RLU) were normalized on total proteins (TP, μg). *, p<0.05; **, p<0.01, ***, p<0.001.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A-D) Representative western blot of (A) lysates from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of PPIA (B) , low-glycosylated (37kDa) (C) and high-glycosylated (50kDa) (D) forms of EMMPRIN (EMN). Data are mean±SEM of n=3 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (E-G) Representative western blot of (E) media from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of extracellular PPIA (ePPIA) (F) and soluble EMMPRIN (sEMN) (G) . Data are mean±SEM of n=3-4 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (H) Luciferase assay for NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 72h and treated with 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3 independent experiments expressed as fold of Mock untreated cells. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: Target protein intensity was normalized on total transferred proteins (TTP). Relative luminescence units (RLU) were normalized on total proteins (TP, μg). *, p<0.05; **, p<0.01, ***, p<0.001.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative Proteomics, Luciferase, Activity Assay, Control

(A) Representative western blot and relative quantification of the high-glycosylated (50kDa) (B) and the low-glycosylated (37kDa) (C) forms of EMMPRIN (EMN) in the ventral horns of the lumbar spinal cord of Ntg (black dots) and SOD1 G93A (red dots) mice. Two-Way Anova: HG-EMN (interaction, p=0.084; age, p<0.0001; genotype, p<0.0001) followed by T-test for genotype comparison: HG-EMN (PS, p=0.4986; ON, **, p=0.0064; SY, ***, p=0.0005; ES, **, p=0.0052); LG-EMN (interaction, p=0.6868; age, p=0.825; genotype, p=0.831). One-Way Anova for linear trend: HG-EMN, p<0.0001; LG-EMN, p=0.0636. Target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=5 mice/stage. (D) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at the onset of the disease. Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. Experiments have been performed in n=3 mice/group. (E) Representative image of EMMPRIN (EMN, gray) expression in astrocytes (GFAP, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. (F) Representative image of EMMPRIN (EMN, gray) expression in microglia (Iba1, red) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. Of note, E and F are the same image but with split channels and performed appropriate merge. (G) Relative quantification of the percentage of astrocytes (GFAP) or microglia (Iba1) expressing EMMPRIN. Two-Way Anova (interaction, p=0.0003; age, p<0.0001; cell type, p<0.0001) followed by Bonferroni multiple comparison test for cell type comparison (***PS, p=0.0001; ON, SY, ES ****, p<0.0001). One-Way Anova for linear trend: GFAP, p<0.0001; Iba1, p<0.0001. Data are mean±SEM of n=4-5 mice/stage. For all experiment: PS, presymptomatic; ON, onset; SY, symptomatic; ES, end-stage. For D-F scale bar = 100μm.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative western blot and relative quantification of the high-glycosylated (50kDa) (B) and the low-glycosylated (37kDa) (C) forms of EMMPRIN (EMN) in the ventral horns of the lumbar spinal cord of Ntg (black dots) and SOD1 G93A (red dots) mice. Two-Way Anova: HG-EMN (interaction, p=0.084; age, p<0.0001; genotype, p<0.0001) followed by T-test for genotype comparison: HG-EMN (PS, p=0.4986; ON, **, p=0.0064; SY, ***, p=0.0005; ES, **, p=0.0052); LG-EMN (interaction, p=0.6868; age, p=0.825; genotype, p=0.831). One-Way Anova for linear trend: HG-EMN, p<0.0001; LG-EMN, p=0.0636. Target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=5 mice/stage. (D) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at the onset of the disease. Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. Experiments have been performed in n=3 mice/group. (E) Representative image of EMMPRIN (EMN, gray) expression in astrocytes (GFAP, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. (F) Representative image of EMMPRIN (EMN, gray) expression in microglia (Iba1, red) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. Of note, E and F are the same image but with split channels and performed appropriate merge. (G) Relative quantification of the percentage of astrocytes (GFAP) or microglia (Iba1) expressing EMMPRIN. Two-Way Anova (interaction, p=0.0003; age, p<0.0001; cell type, p<0.0001) followed by Bonferroni multiple comparison test for cell type comparison (***PS, p=0.0001; ON, SY, ES ****, p<0.0001). One-Way Anova for linear trend: GFAP, p<0.0001; Iba1, p<0.0001. Data are mean±SEM of n=4-5 mice/stage. For all experiment: PS, presymptomatic; ON, onset; SY, symptomatic; ES, end-stage. For D-F scale bar = 100μm.

Techniques: Western Blot, Quantitative Proteomics, Comparison, Expressing, Labeling

(A) Representative image of primary cultures of NTg and SOD1 G93A astrocytes. Most of the cells present in the preparation are astrocytes (GFAP), and only a very small percentage of microglia (Iba1) cells is detected. Scale bar = 100μm. (B) Representative western blot and (C) relative quantification of EMMPRIN (EMN) in NTg astrocytes treated 24h with 0.5nM recombinant PPIA. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05 by unpaired T-Test. (D) Relative quantification of factors released by NTg astrocytes after 24h of treatment with 0.5nM recombinant PPIA (N:P) compared to untreated NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of NTg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 1. (E) Pie chart of upregulated, unchanged or downregulated proteins in NTg astrocytes treated with PPIA compared to untreated NTg astrocytes. (F) Significant leading pathways related to the 43 upregulated proteins found in NTg astrocytes treated with PPIA. Proteins associated with the pathways are listed in Supplementary Table 2.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative image of primary cultures of NTg and SOD1 G93A astrocytes. Most of the cells present in the preparation are astrocytes (GFAP), and only a very small percentage of microglia (Iba1) cells is detected. Scale bar = 100μm. (B) Representative western blot and (C) relative quantification of EMMPRIN (EMN) in NTg astrocytes treated 24h with 0.5nM recombinant PPIA. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05 by unpaired T-Test. (D) Relative quantification of factors released by NTg astrocytes after 24h of treatment with 0.5nM recombinant PPIA (N:P) compared to untreated NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of NTg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 1. (E) Pie chart of upregulated, unchanged or downregulated proteins in NTg astrocytes treated with PPIA compared to untreated NTg astrocytes. (F) Significant leading pathways related to the 43 upregulated proteins found in NTg astrocytes treated with PPIA. Proteins associated with the pathways are listed in Supplementary Table 2.

Techniques: Western Blot, Quantitative Proteomics, Recombinant

Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in NTg and SOD1 G93A astrocytes. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01 by unpaired T-Test. (D) Relative quantification of factors released in 24h conditioned medium from SOD1 G93A astrocytes (G:U) compared to NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 3. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes compared to untreated NTg astrocytes. (F) Venn diagram showing 42 commonly secreted proteins between NTg astrocytes treated with PPIA (N:P) and SOD1 G93A astrocytes untreated (G:U). List of common proteins is present in Supplementary Table 4. (G) Significant leading pathways related to the 62 upregulated proteins found in SOD1 G93A astrocytes. Pathways found also in NTg astrocytes treated with PPIA are labelled with a star. Proteins associated with the pathways are listed in Supplementary Table 5.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in NTg and SOD1 G93A astrocytes. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01 by unpaired T-Test. (D) Relative quantification of factors released in 24h conditioned medium from SOD1 G93A astrocytes (G:U) compared to NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 3. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes compared to untreated NTg astrocytes. (F) Venn diagram showing 42 commonly secreted proteins between NTg astrocytes treated with PPIA (N:P) and SOD1 G93A astrocytes untreated (G:U). List of common proteins is present in Supplementary Table 4. (G) Significant leading pathways related to the 62 upregulated proteins found in SOD1 G93A astrocytes. Pathways found also in NTg astrocytes treated with PPIA are labelled with a star. Proteins associated with the pathways are listed in Supplementary Table 5.

Techniques: Quantitative Proteomics

Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in SOD1 G93A astrocytes treated 24h with 0.5nM anti-EMMPRIN (EMN Ab) or isotype control (CTR Ab) antibodies. Data are mean±SEM of n=3-4 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01, ***, p<0.001 by unpaired T-Test. (D) Relative quantification of the 42 commonly secreted factors between untreated SOD1 G93A and NTg astrocytes treated with PPIA (List in Supplementary Table 4) released from SOD1 G93A astrocytes after 24h treatment with 0.5nM anti-EMMPRIN (G:E) or isotype control (G:C) antibodies. Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=4 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05; ** versus SOD1 G93A astrocytes treated with control antibody by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 6. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes treated with anti-EMMPRIN compared to control antibody treated SOD1 G93A astrocytes. (F) Significant leading pathways related to the 30 downregulated proteins found in SOD1 G93A astrocytes after anti-EMMPRIN treatment. Proteins associated with the pathways are listed in Supplementary Table 7.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in SOD1 G93A astrocytes treated 24h with 0.5nM anti-EMMPRIN (EMN Ab) or isotype control (CTR Ab) antibodies. Data are mean±SEM of n=3-4 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01, ***, p<0.001 by unpaired T-Test. (D) Relative quantification of the 42 commonly secreted factors between untreated SOD1 G93A and NTg astrocytes treated with PPIA (List in Supplementary Table 4) released from SOD1 G93A astrocytes after 24h treatment with 0.5nM anti-EMMPRIN (G:E) or isotype control (G:C) antibodies. Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=4 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05; ** versus SOD1 G93A astrocytes treated with control antibody by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 6. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes treated with anti-EMMPRIN compared to control antibody treated SOD1 G93A astrocytes. (F) Significant leading pathways related to the 30 downregulated proteins found in SOD1 G93A astrocytes after anti-EMMPRIN treatment. Proteins associated with the pathways are listed in Supplementary Table 7.

Techniques: Quantitative Proteomics, Control

(A) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT , TDP-43 A315T , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=6-8 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human TDP-43 WT or TDP-43 A315T and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=3 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT or TDP-43 A315T plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or 0.5nM anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-6 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT , TDP-43 A315T , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=6-8 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human TDP-43 WT or TDP-43 A315T and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=3 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT or TDP-43 A315T plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or 0.5nM anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-6 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Representative dot blot (A) and relative quantification ( B) of extracellular PPIA in the CSF of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. *, p=0.0248. One-Way Anova for linear trend in TDP-43 A315T mice: p=0.1466. Representative western blot ( C ) and relative quantification of the high-glycosylated (50kDa) (D) and the low-glycosylated (37kDa) (E) forms of EMMPRIN (EMN) in the lumbar spinal cord of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. **, p=0.0020; ***, p=0.0001. One-Way Anova for linear trend in TDP-43 A315T mice: HG-EMN, p=0.0051; LG-EMN, p=0.0054. For A-E, target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=3-4 mice/stage (6 months, onset; 10 months, early symptomatic; 13 months, late-symptomatic). (F) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and TDP-43 A315T mice at the onset of the disease (6 months). Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. (G) Representative image of EMMPRIN (EMN, green) expression in astrocytes (GFAP, red) or microglia (Iba1, gray) in the ventral horn of the lumbar spinal cord of TDP-43 A315T mice at the early symptomatic stage (10 months). Please note that due to antigen retrieval we observed some Iba1 leaking signal in neurons (yellow arrow heads). For F, G: experiments have been performed in n=3 mice/group. Scale bar = 100μm.

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Representative dot blot (A) and relative quantification ( B) of extracellular PPIA in the CSF of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. *, p=0.0248. One-Way Anova for linear trend in TDP-43 A315T mice: p=0.1466. Representative western blot ( C ) and relative quantification of the high-glycosylated (50kDa) (D) and the low-glycosylated (37kDa) (E) forms of EMMPRIN (EMN) in the lumbar spinal cord of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. **, p=0.0020; ***, p=0.0001. One-Way Anova for linear trend in TDP-43 A315T mice: HG-EMN, p=0.0051; LG-EMN, p=0.0054. For A-E, target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=3-4 mice/stage (6 months, onset; 10 months, early symptomatic; 13 months, late-symptomatic). (F) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and TDP-43 A315T mice at the onset of the disease (6 months). Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. (G) Representative image of EMMPRIN (EMN, green) expression in astrocytes (GFAP, red) or microglia (Iba1, gray) in the ventral horn of the lumbar spinal cord of TDP-43 A315T mice at the early symptomatic stage (10 months). Please note that due to antigen retrieval we observed some Iba1 leaking signal in neurons (yellow arrow heads). For F, G: experiments have been performed in n=3 mice/group. Scale bar = 100μm.

Techniques: Dot Blot, Quantitative Proteomics, Comparison, Western Blot, Expressing, Labeling

A) Confocal images of perivascular cuffs in the cerebellar white matter of CCR2:EMMP +/+ and CCR2:EMMP −/− D18 EAE showing pan-leukocytic marker, CD45 (red) encased in basement membrane delineated by laminin staining (green). Insets showing enlarged cuffs with CD45+ cells within. Scale-50μm. B) Histograms denoting average number of cuffs observed in the two groups. Data analyzed by student’s T-test. **p<0.05, N of 6 per group. C) Spinal cords were harvested from CCR2:EMMP +/+ and CCR2:EMMP −/− mice at D18 after immunization and subjected to flow cytometry: Singlet viable cells were gated on CD11b and CD45 for CD45hi CD11b+ monocyte/macrophages as shown in dot-plot for CCR2:EMMP −/− and CCR2:EMMP +/+ examples; there were few monocyte/macrophages in the spinal cord of CCR2:EMMP −/− mice. D) Analysis of % EMMPRIN+ CCR2+ infiltrated macrophages as well as E) expression of EMMPRIN levels (MFI = mean fluorescence intensity) in CCR2+ CD11b+LY6G-cells. F) Dot plot exhibiting Ly6G and CD11b staining from blood of CCR2:EMMP +/+ and CCR2:EMMP −/− D12 EAE mice which is quantified as %CD11b+ Ly6G-CD45+ cells in (G) . Histograms showing %CD11b+ Ly6G-CD45+ cells from D18 EAE in WT, CCR2:EMMP +/+ and CCR2:EMMP −/− mice. Flow plots shown in D, E, G and H were compared using one-way ANOVA with Bonferroni post-hoc test. *p<0.05, **p<0.01 and ***p<0.001. Data represented as mean ± SD.

Journal: bioRxiv

Article Title: EMMPRIN confers metabolic advantage for monocytes and macrophages to promote disease in a model of multiple sclerosis

doi: 10.1101/2024.08.11.607460

Figure Lengend Snippet: A) Confocal images of perivascular cuffs in the cerebellar white matter of CCR2:EMMP +/+ and CCR2:EMMP −/− D18 EAE showing pan-leukocytic marker, CD45 (red) encased in basement membrane delineated by laminin staining (green). Insets showing enlarged cuffs with CD45+ cells within. Scale-50μm. B) Histograms denoting average number of cuffs observed in the two groups. Data analyzed by student’s T-test. **p<0.05, N of 6 per group. C) Spinal cords were harvested from CCR2:EMMP +/+ and CCR2:EMMP −/− mice at D18 after immunization and subjected to flow cytometry: Singlet viable cells were gated on CD11b and CD45 for CD45hi CD11b+ monocyte/macrophages as shown in dot-plot for CCR2:EMMP −/− and CCR2:EMMP +/+ examples; there were few monocyte/macrophages in the spinal cord of CCR2:EMMP −/− mice. D) Analysis of % EMMPRIN+ CCR2+ infiltrated macrophages as well as E) expression of EMMPRIN levels (MFI = mean fluorescence intensity) in CCR2+ CD11b+LY6G-cells. F) Dot plot exhibiting Ly6G and CD11b staining from blood of CCR2:EMMP +/+ and CCR2:EMMP −/− D12 EAE mice which is quantified as %CD11b+ Ly6G-CD45+ cells in (G) . Histograms showing %CD11b+ Ly6G-CD45+ cells from D18 EAE in WT, CCR2:EMMP +/+ and CCR2:EMMP −/− mice. Flow plots shown in D, E, G and H were compared using one-way ANOVA with Bonferroni post-hoc test. *p<0.05, **p<0.01 and ***p<0.001. Data represented as mean ± SD.

Article Snippet: Cells were then washed and incubated in following antibodies for 30 minutes at 4°C in the dark: PerCP rat anti-mouse CD45 (557235, BD Biosciences, 1:50), FITC rat anti-CD11b (553310, BD Biosciences, 1:50), APC-Cy7 rat anti-mouse Ly6G (560600, BD Biosciences, 1:50), PE rat anti-mouse EMMPRIN (562676, BD Biosciences, 1:50), BUV395 rat anti-mouse CD115 (564059, BD Biosciences, 1:50), BV510 rat anti-mouse CCR2 (CD192, 747970, BD Biosciences, 1:50) and fixable viability dye eFluor 780 (65-0865-14, eBioscience).

Techniques: Marker, Membrane, Staining, Flow Cytometry, Expressing, Fluorescence

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