Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Enzyme Replacement Therapy Can Reverse Pathogenic Cascade in Pompe Disease

doi: 10.1016/j.omtm.2020.05.026

Figure Lengend Snippet: AT-GAA Reversed the Levels of the Autophagic Markers and Alleviated the Burden of Accumulated Protein Aggregates and ER-Stress in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group; n = 5 for western blot with LC3 antibody). (B) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with K63-linked ubiquitin specific antibody (n = 4 for each group). Western blot with anti-GAPDH and Ponceau S staining were used as loading controls. (C) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with anti-Grp 78 antibody (ER-stress marker; n = 4 for each group); GAPDH was used as loading control. Each data point represents an individual mouse. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: SQSTM1/p62 (#ab56416; mouse monoclonal), galectin 9 (#ab69630; rabbit polyclonal), Ubiquitin K48 (Linkage Specific) (#ab140601; rabbit monoclonal), and glyceraldehyde-3-phosphate dehydrogenase (#ab9485; rabbit polyclonal) were purchased from Abcam.

Techniques: Western Blot, Ubiquitin Proteomics, Staining, Marker, Control

Journal: Frontiers in Oncology

Article Title: Differential O - and Glycosphingolipid Glycosylation in Human Pancreatic Adenocarcinoma Cells With Opposite Morphology and Metastatic Behavior

doi: 10.3389/fonc.2020.00732

Figure Lengend Snippet: Immune recognition of glycan structures on PaTu-S and PaTu-T cells. (A) Interaction of immature DCs with PaTu-S and PaTu-T were visualized by fluorescence microscopy. Bar = 100 μm. (B) Binding of immature DCs to PaTu-S and PaTu-T in a cell adhesion assay, in the presence or absence of EGTA. Results are derived from 6 independent experiments using different donors and expressed as average percentage binding ± SEM. (C) Binding of recombinant human galectins Gal-1, Gal-3, and Gal-4 (5 μg/ml) to the PDAC cell lines was measured by flow cytometry. Results are given as average MFI ± SEM of at least 2 independent experiments. (D) Binding of Fc-chimeras of DC-SIGN, MGL, DCIR and Dectin-1 to PaTu-S and PaTu-T cells was measured by flow cytometry. Results are given as average MFI ± SEM of at least 3 independent experiments. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: Mouse anti-human galectin (Gal)-1 , and anti-Tn monoclonal antibodies were kindly provided by Dr. RD Cummings (Boston, USA), goat anti-human Gal-4 was purchased from R&D Systems (Minneapolis, MN) and anti-sialyl Lewis A/CA 19-9 monoclonal antibody was from LifeSpan Biosciences (Seattle, WA).

Techniques: Fluorescence, Microscopy, Binding Assay, Cell Adhesion Assay, Derivative Assay, Recombinant, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Competition between Core-2 GlcNAc-transferase and ST6GalNAc-transferase Regulates the Synthesis of the Leukocyte Selectin Ligand on Human P-selectin Glycoprotein Ligand-1 *

doi: 10.1074/jbc.M113.463653

Figure Lengend Snippet: PSGL-1 variants. A, shown is mature PSGL-1 with 73 potential sites for O-glycosylation (brown circles) and 3 sites for N-glycosylation (purple squares). N-terminal residues include three sulfated tyrosines (S) and a sialofucosylated O-glycan at Thr-57 (red box). The PSGL-1 variants constructed include either 19 or 76 N-terminal residues followed by the human IgG1 Fc, PSGL-1 transmembrane, and cytoplasmic sections. B, HEK293T and FUT7+HEK cells expressing either full-length PSGL-1 or PSGL-1 variants (19FcTM and 76FcTM) were constructed. Flow cytometry histograms characterize anti-human IgG and anti-PSGL-1 mAb KPL-1 binding (left column), CLA/HECA-452 and CHO-131 epitope expression (middle column), and P-selectin IgG binding (right column). Wild-type HEK293T cells do not express PSGL-1. The CLA epitope is augmented upon FUT7 overexpression. Robust P-selectin IgG binding requires co-expression of FUT7 with either wild-type PSGL-1, 19FcTM, or 76FcTM. Numerical values provided in individual histograms represent mean ± S.D. for 4–7 experiments.

Article Snippet: Mouse monoclonal antibodies used include anti-PSGL-1/CD162 mAb KPL-1, anti-Lewis-X/CD15 mAb HI98, anti-sialyl Lewis-X (sLe X )/CD15s mAb CSLEX-1, anti-CD65s mAb VIM-2 (AbD Serotec, Raleigh, NC), anti-cutaneous lymphocyte associated antigen/CLA mAb HECA-452, and mAb CHO-131 (R&D Systems, Minneapolis, MN).

Techniques: Construct, Expressing, Flow Cytometry, Binding Assay, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Competition between Core-2 GlcNAc-transferase and ST6GalNAc-transferase Regulates the Synthesis of the Leukocyte Selectin Ligand on Human P-selectin Glycoprotein Ligand-1 *

doi: 10.1074/jbc.M113.463653

Figure Lengend Snippet: Overexpression of ST6GalNAc1, -2, and -4 in HL-60. A, lentivirus vector used to overexpress ST6GalNAc-transferases in HL-60 co-expressed a DsRED (red fluorescent protein) reporter. Micrographs show DsRED expression in all transduced HL-60s. IRES, internal ribosome entry site. B, RT-PCR analysis of ST6GalNAc-transferases in wild-type HL-60 and HL-60s overexpressing ST6GalNAc-transferases is shown. RT-PCR products were resolved on an agarose gel and sequence-verified. C, shown is an RT-PCR comparison of ST6GalNAc-transferase expression in HL-60 versus human peripheral blood neutrophils. D, ST6GalNAc-transferase activity in various HL-60 cell lysates was measured by detecting [14C]NeuAc incorporation into α(2,3)-sialylated fetuin substrate. Incorporated radioactivity was released by α(2,3/6/8/9) A. ureafaciens neuraminidase but not α(2,3) M. decora neuraminidase. *, p < 0.001 compared with normal HL-60. E, cell surface expression of PSGL-1 (KPL-1), CD65s/VIM-2, CD15/LeX (HI98), CLA (HECA-452), and CD15s/sLeX (CSLEX-1) on HL-60 and its variants is shown. ST6GalNAc2 and ST6GalNAc4 overexpression reduced CLA/HECA-452 expression with ST6GalNAc2 also reducing the sLeX/CSLEX-1 epitope. Data are the mean ± S.D. for 3–5 experiments. *, p < 0.001 compared with normal HL-60.

Article Snippet: Mouse monoclonal antibodies used include anti-PSGL-1/CD162 mAb KPL-1, anti-Lewis-X/CD15 mAb HI98, anti-sialyl Lewis-X (sLe X )/CD15s mAb CSLEX-1, anti-CD65s mAb VIM-2 (AbD Serotec, Raleigh, NC), anti-cutaneous lymphocyte associated antigen/CLA mAb HECA-452, and mAb CHO-131 (R&D Systems, Minneapolis, MN).

Techniques: Over Expression, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing, Comparison, Activity Assay, Radioactivity