Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Information of primary antibodies used in the present study.

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques:

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Primer sequence and promoter primer sequence.

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: CD147 overexpression correlates with poor prognosis and RAP1 co-expression in colorectal cancer. ( A , B ) CD147 mRNA expression levels in tumor vs. normal tissues across multiple cancers (UCSC Xena database) and specifically in colorectal cancer (CRC) (GEPIA database). ** P < 0.01, unpaired t-test. ( C ) Kaplan-Meier survival analysis of CRC patients stratified by CD147 expression (log-rank test, P < 0.05). ( D ) Representative immunohistochemical images from The Human Protein Atlas showing CD147 expression in normal colorectal tissue (negative) and CRC tissue (cytoplasmic/membrane staining). ( E , F ) Western blot analysis of CD147 protein levels in paired CRC and adjacent normal tissues ( n = 12). β-actin served as loading control. *** P < 0.001, paired t-test. Origin blots are presented in Supplementary Fig. 1. ( G , H ) Correlation analyses between CD147 and RAP1A/B mRNA expression using GEPIA (Pearson’s correlation, P < 0.05) and TIMER2.0 (no significant correlation). ( I ) qRT-PCR validation of CD147 and Rap1 expression correlation in 12 CRC tissues (Pearson’s correlation, P < 0.01).

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Over Expression, Expressing, Immunohistochemical staining, Membrane, Staining, Western Blot, Control, Quantitative RT-PCR, Biomarker Discovery

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: CD147 expression validation and knockdown efficiency in CRC cell lines. ( A ) Immunofluorescence staining of CD147 in SW620 cells showing strong cytoplasmic expression (red) with partial membrane localization. Nuclei were counterstained with Hoechst 33,342 (blue). Scale bar: 30 μm. ( B ) Western blot analysis of CD147 protein levels in HCT116, SW620, and normal colon epithelial HCoEpiC cells. β-actin served as loading control ( n = 3 independent experiments). Original blots are presented in Supplementary Fig. 1. ( C ) qRT-PCR quantification of CD147 mRNA levels normalized to β-actin ( n = 3 independent experiments). ( D - F ) Lentiviral shRNA-mediated CD147 knockdown in HCT116 cells. ( D ) Representative Western blot of CD147 protein levels after transfection with three shRNAs (shCD147-1/2/3) or non-targeting control (shNC). Original blots are presented in Supplementary Fig. 1. ( E ) Densitometric quantification of CD147 protein expression from three independent experiments. ( F ) qRT-PCR analysis of CD147 mRNA levels post-knockdown (normalized to β-actin). Data are mean ± SD; *** P < 0.001 (Student’s t-test).

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Expressing, Biomarker Discovery, Knockdown, Immunofluorescence, Staining, Membrane, Western Blot, Control, Quantitative RT-PCR, shRNA, Transfection

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: CD147 knockdown inhibits proliferation and induces apoptosis in CRC cells. ( A ) CCK-8 proliferation assay showing reduced viability of HCT116 and SW620 cells transfected with shCD147-1 (shCD147) compared to non-targeting control (shNC) at 48 h and 72 h ( n = 3, mean ± SD; *** P < 0.001, one-way ANOVA). ( B , C ) Colony formation assay in shCD147 and shNC groups. ( B ) Representative images of colonies (crystal violet staining). ( C ) Quantification of colony numbers ( n = 3; *** P < 0.001 vs. shNC, Student’s t-test). ( D - F ) Western blot analysis of proliferation/apoptosis-related proteins. ( D ) Representative blots of c-Myc, Bcl-2, and Bax in shCD147 and shNC groups. β-actin served as loading control. Original blots are presented in Supplementary Fig. 2. ( E , F ) Densitometric quantification of protein levels ( n = 3; ** P < 0.01, *** P < 0.001 vs. shNC, Student’s t-test). ( G , H ) Flow cytometry analysis of apoptosis. ( G ) Representative Annexin V-FITC/PI staining profiles. ( H ) Quantification of apoptotic cell percentages ( n = 3; *** P < 0.001 vs. shNC, Student’s t-test).

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Knockdown, CCK-8 Assay, Proliferation Assay, Transfection, Control, Colony Assay, Staining, Western Blot, Flow Cytometry

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: CD147 knockdown suppresses migration, invasion, and epithelial-mesenchymal transition (EMT) in colorectal cancer cells. ( A , B ) Scratch wound healing assay in HCT116 and SW620 cells. ( A ) Representative images of wound closure at 24 h and 48 h post-scratching. ( B ) Quantification of wound closure rate ( n = 3; mean ± SD; ** P < 0.01, *** P < 0.001, vs. shNC, one-way ANOVA). ( C , D ) Transwell migration and invasion assays. ( C ) Representative images of migrated/invaded cells (crystal violet staining). ( D ) Quantification of migrated/invaded cells ( n = 3; *** P < 0.01 vs. shNC, Student’s t-test). ( E , F ) Western blot analysis of EMT-related proteins. ( E ) Representative blots of E-cadherin and N-cadherin in shCD147 and shNC groups. β-actin served as loading control. Original blots are presented in Supplementary Fig. 2. ( F ) Densitometric quantification of protein levels ( n = 3; ** P < 0.01, *** P < 0.001 vs. shNC, Student’s t-test).

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Knockdown, Migration, Wound Healing Assay, Staining, Western Blot, Control

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Rap1 overexpression reverses the expression of key proteins in CD147 knockdown mediated cell proliferation, apoptosis and EMT. ( A , B ) Western blot analysis of Rap1 and Rap1GAP protein levels in HCT116 and SW620 cells after CD147 knockdown (shCD147). β-actin served as a loading control. Data represent mean ± SD ( n = 3; ** P < 0.01, *** P < 0.001 vs. shNC, Student’s t-test). Original blots are presented in Supplementary Fig. 3. ( C ) qRT-PCR validation of Rap1 mRNA overexpression in CD147-knockdown cells. Expression normalized to β-actin ( n = 3; *** P < 0.001 vs. HCT116 or SW620, Student’s t-test). ( D , E ) Western blot analysis of c-Myc, Bcl-2, Bax, N-cadherin, and E-cadherin protein levels in CD147-Knockdown cells with Rap1 overexpression ( n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. shNC, # P < 0.05, ## P < 0.01, ### P < 0.001 vs. shCD147, one-way ANOVA). Original blots are presented in Supplementary Fig. 4.

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Over Expression, Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Biomarker Discovery

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Rap1 overexpression rescues the regulatory effects of CD147 knockdown on proliferation and apoptosis. ( A ) CCK-8 assay showing proliferation of HCT116 ( n = 3; *** P < 0.001 vs. shNC, ### P < 0.001 vs. shCD147, one-way ANOVA). ( B , C ) Colony formation assays in HCT116 cells. Colonies were counted and normalized to shCtrl ( n = 3; ** P < 0.01, *** P < 0.001 vs. shNC, ### P < 0.001 vs. shCD147, one-way ANOVA). ( D , E ) Flow cytometry analysis of apoptosis in HCT116 cells. Percentages of apoptotic cells are shown ( n = 3; * P < 0.05 vs. shNC, # P < 0.05 vs. shCD147, one-way ANOVA). Similar results were observed in SW620 cells.

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Over Expression, Knockdown, CCK-8 Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Rap1 restores CD147-mediated migration and invasion in colorectal cancer cells. ( A , C ) Scratch wound healing assay in HCT116 cells. Healing rates were quantified at 24 h and 48 h ( n = 3; *** P < 0.001 vs. shNC, ## P < 0.01, ### P < 0.001 vs. shCD147, one-way ANOVA). ( B , D ) Transwell migration and Matrigel invasion assays. Migrated/invaded cells were counted and normalized to shCtrl ( n = 3; *** P < 0.001 vs. shNC, ### P < 0.001 vs. shCD147, one-way ANOVA). SW620 cells showed consistent trends. ( E ) Mechanistic diagram of CD147 promoting tumor progression through Rap1/Rap1GAP signaling in colorectal cancer cells.

Article Snippet: Anti CD147 mouse monoclonal antibody , 66443-1-AP , ProteinTech Group, Inc. , 1:5000 /1:200.

Techniques: Migration, Wound Healing Assay

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: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) The upper panel shows the trace from size exclusion chromatography of n -dodecyl-β-D-maltose and cholesterol hemisuccinate (DDM/CHS)-solubilised ghost membrane proteins fractionated on a Superdex 200 increase 10/300 column (blue) and for full-length basigin purified from insect cells in CHAPS and exchanged into DDM:CHS (green). The elution volumes of molecular weight standards are indicated above the trace. Fractions collected are demarcated by vertical red dotted lines. The lower panel shows western blots of fractions 2–14. Representative blots for basigin (40–65 kDa, upper panel), PMCA (~138 kDa, upper middle panel), and MCT1 (~45 kDa, lower middle panel) show that all three proteins co-elute predominantly in fractions 7–9. The lower panel shows the equivalent blot for full-length basigin (FL-BSG), related to the green trace. Data shown are representative from n=2 biological replicates. ( b ) Western blot analysis of 2D blue native PAGE/SDS-PAGE separations of human erythrocyte membrane solubilisates before (upper panel) and after pre-incubation with anti-basigin antibody (lower panel). Blot membranes were stained with antibodies specific for PMCA1/4, basigin and MCT1. Markers of apparent complex size indicate the positions of known mitochondrial respiratory chain (super)complexes run in a separate gel lane. Binding of the antibody led to a full size-shift of both PMCA1/4-basigin and MCT1-basigin complexes, whereas no signal of free basigin could be observed in the low molecular weight range, even after overexposure of the blot. Data shown are representative from n=2 biological replicates. ( c ) The left-hand panel shows representative western blot images depicting sequential depletion of PMCA (upper panel) and MCT1 (upper-middle panel). Depletion of both transporters leads to reduced basigin levels, while levels of glycophorin C are unaffected in each fraction, again confirming that basigin is in complex with PMCA or MCT1. The remaining panels show densitometry plots obtained from inverted images of the western blots. Mean integrated band densities are shown with error bars as the standard error of the mean (n=3) and represent technical replicates. ( d ) Bar diagram depicting molecular abundances (abundance norm spec values) of the indicated proteins in depleting affinity purifications with anti-basigin and anti-neuroplastin (NPTN) antibodies from mildly solubilised human erythrocyte membranes as determined by mass spectrometry. The abundance for all proteins was 0 after affinity purification with an IgG control. Data shown are representative from n=8 biological replicates for basigin and n=5 biological replicates for neuroplastin. Figure 1—source data 1. Data associated with . Figure 1—source data 2. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Size-exclusion Chromatography, Membrane, Purification, Molecular Weight, Western Blot, Blue Native PAGE, SDS Page, Incubation, Staining, Binding Assay, Mass Spectrometry, Affinity Purification, Control

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a, b ) Purification of full-length basigin from baculovirus-infected insect cells was performed in two different detergent mixtures ( n -dodecyl-β-D-maltose and cholesterol hemisuccinate [DDM:CHS] or CHAPS) or was performed in CHAPS, followed by exchange into DDM:CHS (exchanged). ( a ) shows a Coomassie-stained SDS-PAGE analysis of these three samples. ( b ) The left-hand panel shows size exclusion chromatography traces on a S200 10/300 increase column for these samples. The right-hand panel shows surface plasmon resonance measurements of the binding of these three samples (0.4 μM), together with basigin ectodomain (BSGecto, 2 μM) to immobilised P. falciparum reticulocyte homologue 5 (PfRH5) (right). ( c ) Analysis of basigin-containing membrane protein complexes extracted from erythrocytes as in , albeit using a broader mass range resolving blue native gel. Data shown are representative from n=2 biological replicates. ( d ) Repeats of depletion experiments from . ( e ) Assessment of the depletion of basigin from solubilised erythrocyte membranes using beads lacking antibodies, related to . Figure 1—figure supplement 1—source data 1. Data associated with . Figure 1—figure supplement 1—source data 2. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Purification, Infection, Staining, SDS Page, Size-exclusion Chromatography, SPR Assay, Binding Assay, Membrane

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: The top panel shows the outcome of separation of the basigin-rich fraction from n -dodecyl-β-D-maltose and cholesterol hemisuccinate (DDM:CHS)-solubilised ghost membranes on a Superdex 200 increase 10/300 column (blue). This was repeated in the presence of MCT1 antibody (green). Individual fractions are labelled and demarcated using red dashed lines. Above the trace are shown the elution profile of molecular weight standards. The lower panel shows western blots probed with MCT1 (left), basigin (central), and GPC (right) for the fractions from the size exclusion profile, both without (top, -IgG) and with (bottom, +IgG) the MCT1 antibody. This shows mobility shift for both MCT1 and basigin, but not the glycophorin C (GPC) control. Data shown are representative from n=2 technical replicates. Figure 1—figure supplement 2—source data 1. Data associated with . Figure 1—figure supplement 2—source data 2. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Molecular Weight, Western Blot, Mobility Shift, Control

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) A structural model showing a complex of basigin (blue) and PMCA (pink) (based on PDB: 6A69, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. ( b ) A size exclusion profile obtained for calmodulin affinity chromatography-purified PMCA from human erythrocyte ghosts separated on a Superdex 200 increase 10/300 column. The inset shows a western blot probed with an anti-basigin monoclonal antibody indicating co-migration of basigin with PMCA. Data shown are representative from n=3 biological replicates. ( c ) A surface plasmon resonance (SPR) sensogram showing the binding of a concentration series of full-length basigin (twofold dilutions from 4 μM) to immobilised PfRH5. Black lines show data and dashed red lines show fitting to a 1:1 binding model, with a dissociation constant of 0.56 μM. Data shown are representative from n=3 biological replicates. ( d ) An SPR sensogram showing the binding of a concentration series of basigin-PMCA (twofold dilutions from 400 nM) to immobilised PfRH5. Black lines show data and dashed red lines show fitting to a two-state binding model. The derived rate and affinity constants are presented in . Data shown are representative from n=2 biological replicates. ( e ) Activation curves of BK Ca channels recorded in Chinese hamster ovary (CHO) cells expressing BK Ca channels alone (grey), or cells also transfected with human PMCA4b and basigin with (red) and without (black) addition of PfRH5 at 2 μM concentration. Data shown are from n=3 biological replicates and error bars show standard error of mean. ( f ) Concentration-response curves for the inhibition of Ca 2+ -ATPase activity, as determined by measuring inorganic phosphate production, of purified basigin-PMCA complex by known inhibitor caloxin 1c2 (red) and PfRH5 (blue). Each data point represents the mean and error bars represent the standard error of the mean of technical replicates (n=3, technical replicates). Figure 2—source data 1. Data associated with . Figure 2—source data 2. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Affinity Chromatography, Purification, Western Blot, Migration, SPR Assay, Binding Assay, Concentration Assay, Derivative Assay, Activation Assay, Expressing, Transfection, Inhibition, Activity Assay

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) Two-dimensional blue native PAGE western blot analysis as in probed with either anti-PMCA1/4 or anti-basigin. The top panel shows solubilised cells transfected with human PMCA4b and basigin, while the lower panel shows basigin-PMCA4 complex purified from these cells. ( b ) Activation curves of BK Ca channels recorded in Chinese hamster ovary (CHO) cells (grey), or cells also transfected with human PMCA4b and basigin with (red) and without (black) addition of PfRCR at 1 μM concentration. Error bars show standard error of mean from n=5 biological replicates. ( c ) Activation curves of BK Ca channels recorded in CHO cells (grey, in the presence of 100 nM, 1 μM, 5 μM, or 50 μM Ca 2+ ), or cells also transfected with human PMCA4b and basigin with (red) and without (black) addition of PfRCR at 1 μM concentration. Error bars show standard error of mean from n=5 biological replicates. Figure 2—figure supplement 2—source data 1. Data associated with . Figure 2—figure supplement 2—source data 2. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Blue Native PAGE, Western Blot, Transfection, Purification, Activation Assay, Concentration Assay

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) A structural model showing a complex of basigin (blue) and MCT1 (cyan) (based on PDB: 6LYY, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. ( b ) Surface plasmon resonance (SPR) traces after flowing detergent solubilised erythrocyte membrane basigin-rich fractions (red) and membrane fractions depleted for plasma membrane calcium ATPase (PMCA) (pink) and both PMCA and MCT1 (orange) over a PfRH5-coated surface. ( c ) An SPR sensogram showing the binding of a concentration series of basigin-MCT1 (twofold dilutions from 1600 nM) to immobilised PfRH5. Black lines show data and dotted red lines show fitting to a two-state binding model. Data shown are representative from n=3 technical replicates. ( d ) Purified PfRH5 and basigin-MCT1 were assayed for complex formation through size exclusion chromatography using a Superdex 200 increase 10/300 column. The elution profile of basigin-MCT1 alone (blue) and in the presence of PfRH5 (red) are shown. The inset SDS-PAGE gel shows the protein species present in the fractions indicated by stars in the elution trace. ( e ) Growth inhibition assays assessed the effect of antibodies targeting basigin (at 1 μg/ml) and MCT-1 and PMCAs (both at 75 μg/ml) on P. falciparum growth in human blood culture. Five mM EDTA was used as a positive control (100% growth inhibition) while complete media was used as a negative control (0% growth inhibition). Data are the mean and standard deviation of three technical replicates, with a single example from three biological repeats shown. Figure 3—source data 1. Data associated with . Figure 3—source data 2. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: SPR Assay, Membrane, Clinical Proteomics, Binding Assay, Concentration Assay, Purification, Size-exclusion Chromatography, SDS Page, Inhibition, Positive Control, Negative Control, Standard Deviation

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: Human erythrocytes were stained with monoclonal antibodies targeting the extracellular domains of MCT1, PMCA, or basigin. Western blots are for gels loaded with 15 μg of sample and are probed with anti-MCT1 (top-left, 1:250 dilution), anti-PMCAs (top-right, 1:500 dilution), and anti-basigin (bottom-right, 1:1000 dilution), Experiments were done twice (n=2). Figure 3—figure supplement 2—source data 1. Gels and blots associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Staining, Bioprocessing, Western Blot

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) The impact of five different PfRH5-binding monoclonal antibodies on the binding of PfRH5 to basigin ectodomain. Data plot shows mean and standard error from three biological replicates (n=3). ( b ) Assessment of the impact of the same monoclonal antibodies binding to full-length basigin (left), basigin-PMCA complex (centre), and basigin-MCT1 complex (right) by surface plasmon resonance analysis. ( c ) Analysis of the dose response of inhibition of binding of basigin-PMCA to immobilised PfRH5 with different concentrations of R5.016. The left-hand panel shows surface plasmon resonance traces while the right-hand panel shows the response plotted against R5.016 concentration. Figure 4—figure supplement 1—source data 1. Data associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Binding Assay, Bioprocessing, SPR Assay, Inhibition, Concentration Assay

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: Five different PfRH5-binding monoclonal antibodies were tested for the inhibition of the binding of PfRH5 to ( a ) full-length basigin, ( b ) basigin-PMCA complex, and ( c ) basigin-MCT1 complex. In each case data (n=3) shown are the mean and error bars represent standard error of mean of biological replicates for full-length basigin and basigin-PMCA and technical replicates for basigin-MCT1. ( d ) Structural models showing a complex of basigin (blue) and PMCA (pink) (based on PDB: 6A69, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. Onto this model has been docked either the complex of PfRH5 (yellow) bound to the Fab fragment of R5.016 (red, PDB:6RCV, ) or of 9AD4 (green; 4U0R, ). ( e ) Structural models showing a complex of basigin (blue) and MCT1 (cyan) (based on PDB: 6LYY, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. Onto this model has been docked either the complex of PfRH5 (yellow) bound to the Fab fragment of R5.016 (red, PDB:6RCV, ) or of 9AD4 (green; 4U0R, ). Figure 4—source data 1. Data associated with .

Article Snippet: 600 μl of the solubilisate was mixed with anti-basigin antibodies (8.4 μg, TA501164 [9H5]+TA501189 [10E10], both Origene) for 2 hr and 600 μl remained untreated.

Techniques: Binding Assay, Bioprocessing, Inhibition

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: Fluorescent micrographs of HEK293 cells transfected with (A, B) SAM-6 μHC and SAM-6 λLC construct pair, (C, D) SAM-6 μHC subunit alone, or (E, F) SAM-6 λLC subunit alone. On day-2 post-transfection, transfected cells were seeded onto poly-D-lysine coated coverslips in the absence (A, C, E) or presence (B, D, F) of 15 μg/mL Brefeldin A (BFA) and cultured statically for 24 hr. On day-3 post-transfection, cells were fixed, permeabilized, and immunostained. (A, B) Co-staining was performed using FITC-labeled anti-human μHC antibody and Texas Red-labeled anti-human λLC antibody. (C, D) Co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. (E, F) Co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to make ‘overlay’ views in A and B. DIC and red image fields were superimposed to create ‘overlay’ views in C‒F.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Transfection, Construct, Cell Culture, Staining, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A) Schematic representation of SAM-6 λLC subunit (top), SAM-6 μHC subunit (middle), and SAM-6 μHC-ΔCH1 mutant which lacks the CH1 domain (bottom). The deleted CH1 domain is shown as a dotted line. Individual domain names are indicated in each box. ER targeting is driven by a heterologous signal sequence adapted from a VK1 encoding gene. (B) Fluorescent micrographs of HEK293 cells transfected with μHC (first row) or μHC-ΔCH1 mutant (second and third rows). On day-3 post-transfection, cells were fixed, permeabilized, and immunostained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views. (C) Fluorescent micrographs of HEK293 cells transfected with [λLC + μHC] pair (first row) or [λLC + μHC-ΔCH1] pair (second and third rows). Immunostaining was performed using FITC-labeled anti-human μHC antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to create ‘overlay’ views. (D‒G) HEK293 cells were transfected with μHC (lanes 1 and 5), μHC-ΔCH1 (lanes 2 and 6), [λLC + μHC] pair (lanes 3 and 7), or [λLC + μHC-ΔCH1] pair (lanes 4 and 8). Cell culture media were harvested at day-7 post-transfection and analyzed by SDS-PAGE under reducing conditions (D, E; lanes 1‒4) or non-reducing conditions (F, G; lanes 1‒4). Cell lysate samples were also prepared on day-7 post-transfection and analyzed by SDS-PAGE (D, lanes 5‒8) or Western blotting (E, lanes 5‒8) after resolving the proteins under reducing conditions. Western blotting was performed using polyclonal anti-IgM (H+L) to detect both μHC and λLC subunits simultaneously as well as assembly intermediates composed of μHC or λLC or both. A faintly detectable μHC-ΔCH1 is pointed by a black arrowhead (E, lane 4). Likewise, faintly detectable μHC-ΔCH1 covalent dimers are pointed by black arrowhead (G, lane 4). The assembled hexameric IgM product is pointed by a red arrowhead (F, G; lane 3). Identifiable assembly intermediates are labeled next to the corresponding bands in panels F and G.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Sequencing, Transfection, Labeling, Immunostaining, Cell Culture, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A, top) Schematic representation of the full-length SAM-6 λLC (top row) and its ΔCS mutant (second row) in which two C-terminal amino acids (Cys-213 and Ser-214) are deleted. (A, bottom) The position of Cys-213 residue involved in the HC‒LC inter-chain disulfide bond is highlighted in yellow in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (B, C) HEK293 cells were transfected with full-length λLC (lanes 1, 3, 5) or its ΔCS mutant (lanes 2, 4, 6). At day-7 post-transfection, cell lysates (lanes 1 and 2) and cell culture media samples (lanes 3 and 4) were prepared and resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (B) or by Western blotting (C). The day-7 cell culture media were also analyzed by Coomassie staining or Western blotting after resolving the proteins under non-reducing conditions (B and C, lanes 5 and 6). Blotted membranes were probed with polyclonal anti-λLC antibodies. The corresponding protein band for the λLC subunit is pointed by an arrowhead and labeled (lanes 1‒4). Monomeric and dimeric free λLC subunit is labeled next to lane 6. (D, E) Fluorescent micrographs of HEK293 cells transfected with full-length λLC (D) or ΔCS mutant (E). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Residue, Transfection, Cell Culture, SDS Page, Staining, Western Blot, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A, left) Schematic representation of parental SAM-6 μHC (top row) and its C137S and C337S mutants (second and third rows). (A, right) The position of Cys-137 and Cys-337 residues is highlighted in yellow in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (B, C) HEK293 cells were transfected with parental μHC and its mutants, as shown at the top of each lane. At day-7 post-transfection, cell culture media (B) and cell lysates (C) were prepared and resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (B, C, left panels) or by Western blotting (B, C, right panels). Blotted membranes in B and C were probed with polyclonal anti-IgM (H+L) antibodies. Both parental and mutant μHCs failed to secrete to the culture media. (D) Fluorescent micrographs of HEK293 cells transfected with parental μHC (top row), μHC (C137S) mutant (second and third rows), or μHC (C337S) mutant (fourth and fifth rows). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Transfection, Cell Culture, SDS Page, Staining, Western Blot, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A) Schematic representation of parental SAM-6 μHC (top row) and its 4×C>S mutant (second row). The positions of key cysteine residues important for the inter-chain disulfide formation are marked on the parental μHC. (B) The position of all four Cys residues involved in inter-chain disulfide bond formation on μHC and Cys-213 on λLC are depicted in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (C, D) HEK293 cells were transfected with parental μHC alone (lane 1) or its 4×C>S mutant alone (lane 2). Likewise, the cells are co-transfected with μHC and JC (lane 3) or μHC (4×C>S) and JC (lane 4). At day-7 post-transfection, cell lysates (C) and culture media (D) were resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (C, D; left panel) or by Western blotting (C, D; right panels). Blotted membranes in C and D were probed with polyclonal anti-IgM (H+L) antibody (top panel) or monoclonal anti-JC antibody (bottom panel). (E) Day-7 culture media were also analyzed by Western blotting after proteins were resolved under non-reducing conditions. Blotted membranes were probed with polyclonal anti-IgM (H+L) antibody (left panel) or monoclonal anti-JC antibody (right panel). (F) Fluorescent micrographs of HEK293 cells transfected with parental μHC (top row) or μHC (4×C>S) mutant (second and third rows). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views. (G) Fluorescent micrographs of HEK293 cells co-transfected with μHC and JC (top row) or μHC (4×C>S) mutant and JC (second and third rows). Cells were co-stained FITC-labeled anti-human μHC (shown in green) and monoclonal anti-JC antibody followed by AlexaFluor594-conjugated secondary antibody (shown in red). Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Transfection, SDS Page, Staining, Western Blot, Labeling