rat anti mouse emmprin  (R&D Systems)

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

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.

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

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.

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

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.

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: Western Blot, Quantitative Proteomics, Recombinant

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.

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: Quantitative Proteomics

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.

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: Quantitative Proteomics, Control

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.

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

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.

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

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

Journal: Frontiers in Immunology

Article Title: Active Vaccination With EMMPRIN-Derived Multiple Antigenic Peptide (161-MAP) Reduces Angiogenesis in a Dextran Sodium Sulfate (DSS)-Induced Colitis Model

doi: 10.3389/fimmu.2018.02919

Figure Lengend Snippet: 161-MAP active vaccination reduces EMMPRIN expression in the colon. (A) Representative images of colon sections stained for EMMPRIN and their quantification using the H-score ( n = 5 per group). Scale bar is 25 μm (B) Representative image of fluorescently labeled EMMPRIN (red) and macrophages (green). Scale bar is 100 μm. (C) Determination of EMMPRIN concentrations in colon lysates ( n = 9–10 per group), and in serum samples ( n = 8–9 per group) by ELISA.

Article Snippet: Endogenous peroxidase was quenched in 3% H 2 O 2 solution for 10 min, slides were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: rat anti-mouse EMMPRIN (R&D systems, MAB772, Minneapolis, MN, USA) diluted 1:250; rat monoclonal anti-F4/80 (Abcam, ab6640, Cambridge, UK) diluted 1:200; rabbit polyclonal anti-CD8 (Bioss, bs-0648R, Woburn, MA, USA) diluted 1:400.

Techniques: Expressing, Staining, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Active Vaccination With EMMPRIN-Derived Multiple Antigenic Peptide (161-MAP) Reduces Angiogenesis in a Dextran Sodium Sulfate (DSS)-Induced Colitis Model

doi: 10.3389/fimmu.2018.02919

Figure Lengend Snippet: 161-MAP active vaccination reduces angiogenesis. (A) Colon sections were stained for CD31 and the vessel surface area was calculated ( n = 4 per group). Scale bar is 100 μm. (B) Concentrations of VEGF and MMP-9 were determined in serum sample by ELISA, and in the colon lysates, normalized to the total protein amounts ( n = 9 per group). (C) Wound scratch assay: colon lysates (25 μg of total protein) were diluted (1:4) and applied onto a confluent layer of the mouse bEND3 endothelial cells (10 5 cells/ 96-plate well) that was scratched with a toothpick. Images were acquired at the beginning of the experiment (T0) and at the end after 24h (T24). The migration area was calculated by subtracting the area of the wound at T24, after endothelial cell migrated and partially closed the wound, from the area of the wound at T0. An EMMPRIN specific blocking antibody (161-pAb) was added to some of the wells as indicated. ( n = 9–10 for the male mice, n = 8 for the female mice). Magnification is x4.

Article Snippet: Endogenous peroxidase was quenched in 3% H 2 O 2 solution for 10 min, slides were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: rat anti-mouse EMMPRIN (R&D systems, MAB772, Minneapolis, MN, USA) diluted 1:250; rat monoclonal anti-F4/80 (Abcam, ab6640, Cambridge, UK) diluted 1:200; rabbit polyclonal anti-CD8 (Bioss, bs-0648R, Woburn, MA, USA) diluted 1:400.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Migration, Blocking Assay