anti cosmc  (Proteintech)

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Schematic of mucin-type O -GalNAcylation. Polypeptide GalNAc-transferases (ppGalNAc-Ts/GALNTs; ∼20 isoenzymes) initiate addition of GalNAc to serine or threonine residues of core proteins. Loss of COSMC or C1GALT1 blocks core 1 extension (T antigen), resulting in accumulation of truncated O -glycans (Tn antigen). VVA lectin detects Tn antigen, whereas PNA lectin detects T antigen. Western blot validation of (B) COSMC -/- and (C) C1GALT1 -/- knockout cell lines; GAPDH serves as a loading control. (D-E) Flow cytometry analysis of VVA and PNA lectin to wild-type and COSMC/C1GALT1 knockout cells. (F-G) LC-MS quantification of total cell surface heparan sulfate (HS) and chondroitin sulfate (CS) levels. (H) Schematic of GAG-specific antibodies: 3G10 recognizes HS stubs generated by heparin lyase and 2B6 detects CS stubs generated by chondroitinase ABC. (I) Flow cytometry analysis of 3G10 binding in wild-type and knockout cells. (J) Flow cytometry analysis of 2B6 binding in wild-type and knockout cells. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with ****p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Western Blot, Biomarker Discovery, Knock-Out, Control, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy, Generated, Binding Assay

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: CRISPR sgRNA targeting of human (A) COSMC and (B) C1GALT1 in TC28a2 chondrocytes. Frameshift biallelic mutations in clonal cell lines for Exon 1 of COSMC and Exon 2 of C1GALT1 were confirmed by Sanger sequencing. (C) Flow cytometry analysis of anti-Tn antibody binding to wild-type and COSMC/C1GALT1 knockout cells (n = 3 independent experiments). (D) Workflow for isolation, purification, enzymatic digestion, aniline tagging, and LC-MS analysis of glycosaminoglycans. GRIL-LC-MS analysis of (E) HS disaccharides, (F) HS sulfates per disaccharide, and (G) HS sulfation in wild-type, COSMC -/- , and C1GALT1 -/- knockout lines (n = 4 independent experiments). GRIL-LC-MS analysis of (H) CS/DS disaccharides and (I) CS/DS sulfation of wild-type and knockout cells (n ≥ 3 independent experiments). Data points are shown as mean ± SD; p-values were determined by student t-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: CRISPR, Sequencing, Flow Cytometry, Binding Assay, Knock-Out, Isolation, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Diagram of the FGF-FGFR-heparan sulfate ternary complex, whch regulates downstream pathways including MAPK/ERK signaling. (B-C) FGF1 and FGF2 ligand binding were significantly reduced in knockout cells versus wild-type control cells. (D) Cell surface expression of FGFR1 was unchanged in wild-type and knockout cells, as quantified via flow cytometry. (E) Western blot analysis of FGF2 induced ERK phosphorylation (p-ERK) and total ERK (t-ERK) levels in wild-type and knockout cells. (F) Western blot band intensities were quantitated by densitometry and plotted as the ratio of p-ERK to t-ERK signal. (G) Western blot analysis of C1GALT1 protein levels upon reintroduction of human C1GALT1 (hC1GALT1) into C1GALT1 -/- cells. (H) Flow cytometry analysis of FGF2 binding in wild-type, C1GALT1 -/- , and C1GALT1 -/- cells transduced with C1GALT1 cDNA. (I) Flow cytometry analysis of HS stubs using 3G10 antibody, and FGF binding in A20D- COSMC-CDG patient fibroblasts. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test (A-H) and student’s T-tests (I) with ****p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Ligand Binding Assay, Knock-Out, Control, Expressing, Flow Cytometry, Western Blot, Phospho-proteomics, Binding Assay, Transduction

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: Validation of CRISPR targeting for additional (A) COSMC (COSMC c6 ) and (B) C1GALT1 (C1GALT1 t2 ) knockout clones. Frameshift biallelic mutations were confirmed by Sanger sequencing. Flow cytometry analysis of (C) PNA lectin, (D) VVA lectin, (E) anti-Tn antibody, (F) 3G10, (G) FGF1, and (H) FGF2 binding in COSMC c6 and C1GALT1 t2 knockout clones compared with wild-type control cells. Data points are shown as mean ± SD (n ≥ 3independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Biomarker Discovery, CRISPR, Knock-Out, Clone Assay, Sequencing, Flow Cytometry, Binding Assay, Control

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: Venn diagrams comparing significantly downregulated (A) and upregulated (B) genes (Fold change ≥ 2, FDR p ≤ 0.01) from RNA-seq datasets for COSMC -/- and C1GALT1 -/- knockout cells. Gene Ontology enrichment analysis of overlapping 128 downregulated (C) and 219 upregulated (D) genes shared in COSMC -/- and C1GALT1 -/- cells. (E) Differential expression analysis of proteoglycan and GAG biosynthetic genes (FDR p ≤ 0.01) compared to wild-type control cells. Flow cytometry analysis of (F) syndecan-1 (SDC1) and (G) syndecan-2 levels in TC28a2 wild-type, COSMC -/- , and C1GALT1 -/- cells. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: RNA Sequencing, Knock-Out, Quantitative Proteomics, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) PCA analysis reveals distinct clustering of TC28a2 wild-type, COSMC -/- , and C1GALT1 -/- triplicate samples. (B) Hierarchical clustering and differential expression analysis of triplicate RNA-seq datasets. (C) qPCR validation of gene expression changes in additional COSMC c6 and C1GALT1 t2 knockout clones. Data points are shown as mean ± SD (n = 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Quantitative Proteomics, RNA Sequencing, Biomarker Discovery, Gene Expression, Knock-Out, Clone Assay

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Commasie stained SDS-PAGE gel for purified recombinant human ST6GALNAC1-GFP fusion protein. (B) Schematic of one-step SEEL labeling of TC28a2 cells with ST6GALNAC1 and CMP-Neu5Ac-C5-triazole-biotin. (C) Representative western blot showing streptavidin enrichment versus flow through from SEEL labeling of COSMC -/- and C1GALT1 -/- cells. The one-step SEEL reactions with ST6GALNAC1 were performed as described in the Methods section. (D) The SEEL labeling method detected a number of known O -GalNAcylated glycoproteins (black), including multiple heparan sulfate proteoglycans (blue).

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Staining, SDS Page, Purification, Recombinant, Labeling, Western Blot

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Workflow for analysis of the secretome from TC28a2 wild-type, COSMC -/- , and C1GALT1 -/- cells. (B) Venn diagram showing the overlapping protein hits identified in the secretome analysis of COSMC -/- and C1GALT1 -/- cells versus wild-type controls (n = 3 independent experiments). Over half of the identified protein hits were shared between the knockout cell lines. Volcano plots showing proteins significantly altered in abundance in (C) COSMC -/- and (D) C1GALT1 -/- cells versus wild-type controls. Proteins were categorized and highlighted as Proteoglycans , O-GalNAcylated proteins , Proteases , ECM remodeling enzymes , Growth factors/cytokines/signaling proteins , and other proteins . p-values were determined by Student t-test, and significantly altered proteins were identified using a threshold of p ≤ 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Time course quantification of cell surface SDC1 levels via flow cytometry prior to and after trypsinization (0-24 hours). Data is normalized to untreated wild-type cells (dashed line). (B) Quantification of SDC1 recovery to the cell surface. Data were normalized to the respective untreated control for each genotype and fitted using linear regression. Table inset includes slopes ± SD for each. Statistical comparisons were performed using an F-test to compare the slopes of knockout lines versus wild-type cells (* p < 0.05). (C) Quantification of total SDC1 protein levels in wild-type, COSMC -/- , and C1GALT1 -/- cells treated with bafilomycin A1 (0.25 µM) or DMSO vehicle for 16 hours. (D) Representative histograms from flow cytometry analysis of total SDC1 levels in cells treated with bafilomycin A1. (E) Flow cytometry analysis of cell surface SDC1 levels after treatment with bafilomycin A1 or DMSO vehicle. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test (A,C), and student’s T-test (E) with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Flow Cytometry, Control, Knock-Out

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Schematic representation of CD44 protein structure. CD44 is extensively O -glycosylated, and alternative splicing generates multiple isoforms, with CD44v3 bearing a heparan sulfate chain that can mediate FGF binding. (B) Western blotting and (C) quantification of CD44 protein levels using a pan-CD44 antibody in TC28a2 wild-type, COSMC -/- , and C1GALT1 -/- cells, normalized to GAPDH (n = 3 independent experiments). (D) Reintroduction of hC1GALT1 restored cell surface CD44 levels in C1GALT1 -/- knockout cells. (E) Detection and quantification of cell surface CD44v3 levels in wild-type and mutant lines using an anti-CD44v3 antibody and flow cytometry. (F) Validation of TC28a2 CD44 -/- knockout cells via Western blot using a pan-CD44 antibody. Flow cytometry analyses of cell surface CD44v3 levels, (H) 3G10 binding, and (I) FGF1 and (J) FGF2 binding in wild-type and CD44 -/- cells. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test (C-E), and student’s T-test (G-J) with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Alternative Splicing, Binding Assay, Western Blot, Knock-Out, Mutagenesis, Flow Cytometry, Biomarker Discovery

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: (A) Flow cytometry analysis of cell surface CD44 levels in COSMC c6 and C1GALT1 t2 knockout clones. (B) Time course quantification of cell surface CD44 levels via flow cytometry prior to and after trypsinization (0-24 hours). Data is normalized to untreated wild-type cells. Data points are shown as mean ± SD (n = 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. (C) CRISPR sgRNA targeting of human CD44 in TC28a2 chondrocytes. (C) Frameshift biallelic mutations in a clonal cell line for Exon 5 of CD44 were confirmed by Sanger sequencing.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Flow Cytometry, Knock-Out, Clone Assay, CRISPR, Sequencing

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: Flow cytometry analysis of (A) VVA lectin, (B) PNA lectin, (C) 3G10 antibody, (D) FGF1, (E) FGF2, and (F) pan-CD44 antibody binding in wild-type and knockout growth-plate-like chondroprogenitors (GPLCs). (G) Micromass cultures of GPLC wild-type, Cosmc -/- , and C1galt1 -/- cells at day 4 and day 7 after chondrogenic induction with insulin-transferrin-selenium (ITS). At each time point, cultures were fixed and stained with Alcian blue. (H) Quantification of Alcian blue staining confirmed visual data and confirmed statistical significance at Day 7. qPCR analysis of chondrogenic markers (I) Acan (J) Col2a1 , and (K) Sox9 in wild-type and knockout GPLC micromass cultures at Day 7. Data points are shown as mean ± SD (n ≥ 3 independent experiments); p-values were determined by one-way ANOVA with Tukey post-test with **** p < 0.0001, *** p < 0.001, ** p < 0.01.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: Flow Cytometry, Binding Assay, Knock-Out, Staining

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: CRISPR sgRNA targeting of mouse (A) Cosmc and (B) C1galt1 in murine growth plate-like chondroprogenitors (GPLCs). Frameshift biallelic mutations in a clonal cell line for Exon 1 of Cosmc and Exon 2 of C1galt1 were confirmed by Sanger sequencing.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques: CRISPR, Sequencing

Journal: bioRxiv

Article Title: Mucin-type O -glycans regulate proteoglycan stability and chondrocyte maturation

doi: 10.64898/2025.12.11.693745

Figure Lengend Snippet: Proposed model of proteoglycan homeostasis and ECM remodeling alterations caused by truncation of O -GalNAc glycans in COSMC/C1GALT1 -deficient cells.

Article Snippet: Membranes were probed with primary antibodies against COSMC (Proteintech, #19254-1-AP; 1:1,000), C1GALT1 (Santa Cruz Biotechnology, #sc-100745; 1:500), CD44 (Cell Signaling Technology, #3570; 1:1000), p44/42 MAPK (ERK1/2; Cell Signaling Technology, #9107; 1:1000), phospho-p44/42 MAPK (p-ERK; Cell Signaling Technology, #4370, 1:2000), GAPDH (Cell Signaling Technology, #5174; 1:2500), or β-actin (Cell Signaling Technology, #3700; 1:5000) at 4°C overnight.

Techniques:

Journal: Journal of inherited metabolic disease

Article Title: C1GALT1C1-Associated Mosaic Disorder of Glycosylation in a Female.

doi: 10.1002/jimd.70006

Figure Lengend Snippet: FIGURE 3 | Cosmc-R68 patient leukocytes exhibit reduced Cosmc and T-synthase expression and function. (A) Whole-cell lysates prepared from healthy and patient leukocytes were analyzed on SDS-PAGE western blot and probed for endogenous Cosmc and T-synthase as indicated on the right. Beta-actin serves as the loading control for the respective blots. All images are representative examples of n = 3. (B) ImageJ quantification of the Cosmc blot in Panel A, normalized with respective beta-actin, n = 3, an average of 3, ±1 SD. (C) ImageJ quantification of the T-synthase blot in Panel A, normalized to the respective beta-actin, n = 3, an average of 3, ±1 SD. (D) Similar to (A), whole-cell lysates from healthy and Cosmc-R68 leukocytes were analyzed to measure the T-synthase activity as indicated at the bottom. n = 7, two independent experiments of n = 3 and 4, an average of 7, ±1 SD. (E) Schematics of WT Cosmc and Cosmc-R68 with an N-terminus HA tag. (F) HA-Cosmc-R68 shows truncated expression of the Cosmc protein. Whole-cell lysates prepared from transiently transfected HA-Cosmc and HA-Cosmc-R68 in the CosmcKO cell line or mock were analyzed by SDS-PAGE-WB and probed as indicated on the right. HEK cells were used as a WT control. The arrow shows the HA-tagged expressed Cosmc proteins. n = 3, a representative example of three independent experiments. Beta-actin serves as a loading control (all images are representative ex- amples of n = 3). (G) Parallel to (F), freshly prepared lysates were measured for T-synthase activity. n = 12, three independent experiments of n = 4, an average of 12, ±1 SD.

Article Snippet: Membranes were incubated overnight with primary antibodies prepared in 1× TBST containing 5% milk, including anti- Cosmc (mouse, H10, Santa Cruz #SC- 271829, 1:2000), anti- T- synthase (mouse, F31, Santa Cruz #SC- 100745, 1:2000), anti- β- actin (HRPconjugated, Sigma- Aldrich A3854, 1:10000), and anti- HA (rabbit, Cell Signaling, HA- Tag (C29F4) mAb #3724, 1:1000 in 5% BSA in 1× TBST).

Techniques: Expressing, SDS Page, Western Blot, Control, Activity Assay, Transfection

Journal: Journal of inherited metabolic disease

Article Title: C1GALT1C1-Associated Mosaic Disorder of Glycosylation in a Female.

doi: 10.1002/jimd.70006

Figure Lengend Snippet: FIGURE 4 | Characterization of glycoproteins from Cosmc-R68 patient serum and RBCs. (A) Glycan structures and their respective interacting lectins and glycosidases are shown. The red arrow indicates the digestion sites within the glycan structure where the glycosidases release the gly- cans. Glycan symbols are shown at the bottom. (B) Analysis of serum glycoproteins. Diluted serum samples were treated with neuraminidase (Neu A, removes sialic acid), Neu A + O-glycosidase (removes non-sialylated T-antigen), PNGase-F (removes all N-glycans), or mock-treated. Samples were analyzed on SDS-PAGE-lectin blot (LB) probed with PNA (binds to normal non sialylated O-glycan, T-antigen). Ponceau staining serves as a loading control. LB images are representative examples of four independent experiments. (C) ImageJ quantification of LB from (B) (only Lanes 2 and 6 were quantified, and whole lanes were used in quantification). n = 4, average of 4, ±1 SD. Fold change in total signal intensity is reported. (D) Similar to (B), samples were processed and analyzed on SDS-PAGE-LB and probed with VVA (binds to truncated O-glycans, Tn-antigen). Ponceau staining serves as a loading control. An asterisk (*) on the LB indicates nonspecific binding. LB images are representative examples of four independent experiments. (E) ImageJ quantification of LB from (D) (only Lanes 3 and 7 included; the arrow shows the area used in quantification). n = 4, average of 4, ±1 SD. Fold change in total signal intensity is reported. The dashed line below indicates the background signal of VVA. (F, G) Similar to (B) and (D), samples were processed and analyzed on SDS-PAGE-LB probed with ConA lectin (binds to mannosylated N-glycans) as shown in (F) and secondary antibody alone in (G). (H) Analysis of whole RBC lysate for extended O-glycans. RBC lysates were processed with glycosidases as defined in (B) and analyzed using SDS-PAGE-LB and probed with PNA. LB image is a representative example of four independent experiments. (I) ImageJ quantification of LB from (H) (only lanes with neuraminidase-treated samples included; the arrow shows the area covered in quantification). n = 4, average of 4, ±1 SD. Normalized intensity of PNA with respective beta-Actin intensity is reported.

Article Snippet: Membranes were incubated overnight with primary antibodies prepared in 1× TBST containing 5% milk, including anti- Cosmc (mouse, H10, Santa Cruz #SC- 271829, 1:2000), anti- T- synthase (mouse, F31, Santa Cruz #SC- 100745, 1:2000), anti- β- actin (HRPconjugated, Sigma- Aldrich A3854, 1:10000), and anti- HA (rabbit, Cell Signaling, HA- Tag (C29F4) mAb #3724, 1:1000 in 5% BSA in 1× TBST).

Techniques: Glycoproteomics, SDS Page, Staining, Control, Binding Assay