anti muc13  (Novus Biologicals)

Journal: Nature Communications

Article Title: Polystyrene nanoplastics disrupt the intestinal microenvironment by altering bacteria-host interactions through extracellular vesicle-delivered microRNAs

doi: 10.1038/s41467-025-59884-y

Figure Lengend Snippet: A Alcian blue-periodic acid Schiff (AB-PAS) staining of intestinal mucus in mice with or without NP treatment. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mea n ± SEM ( n = 12). B IHC stain of intestinal MUC-13 in NP-treated mice. Scale bars indicate 100 µm (upper) and 50 µm (lower). Data were shown as mea n ± SEM ( n = 12) (*** p value < 0.001). C MUC13 expression in NP-treated enterocyte-like differentiated Caco-2 cells for 48 h by ICC stain (Scale bars indicate 20 µm), qPCR, and Western blot. Data are presented as means ± SD ( n = 3). Significant difference was shown by different letters (* p < 0.05; *** p < 0.001). D Western blot analysis of MUC-13 levels in goblet-like LS174T cells treated with NP for 48 h. E Heatmap predicting various intestinal miRNAs suppressing MUC-13 in NP-exposed mice. Validation of miR-700-5p interference on MUC-13 in enterocyte-like differentiated Caco-2 cells by ( F ) qPCR. G ICC stain (Scale bars indicate 20 µm), and ( H ) Western blot. Data are presented as means ± SD ( n = 3). Significant difference was shown by different letters (*** p < 0.001). I Schematic of NP im p act on MUC-13 and mucus secretion in the gut.

Article Snippet: For Western blot, the primary antibodies for beta-actin (SC-47778, Santa Cruz, Dallas, Texas, USA), MUC13 (bs-10074R, Bioss, Boston, Massachusetts, USA), GAPDH (GTX100118, GENETEX, Texas, USA), ZO-1 (ab96587, Abcam, Bristol, UK), and OCC (ab216327, Abcam, Bristol, UK) were used to evaluated protein level.

Techniques: Staining, Expressing, Western Blot, Biomarker Discovery

Journal: Nature Communications

Article Title: Polystyrene nanoplastics disrupt the intestinal microenvironment by altering bacteria-host interactions through extracellular vesicle-delivered microRNAs

doi: 10.1038/s41467-025-59884-y

Figure Lengend Snippet: A The effects of NP treatment (1 × 10 10 particles/mL) on the growth of various lactic acid bacteria ( L. paracasei, L. acidophilus , and P. acidiloctici ) , Lachnospiraceae sp. (TSD-26; ATCC), and Ruminococcaceae sp. (TSD-27; ATCC). B Schematic of experimental process by interactions between bacterial EV and cell-derived EV. C Impact of Lachnospiraceae sp.-derived EV without or with NP treatment (1 × 10 10 particles/mL) for 18 h on the growth of different bacterial species ( L. paracasei , L. acidophilus , P. acidiloctici , and Ruminococcaceae sp.). D The impact of Ruminococcaceae sp.-derived EV without or with NP treatment (1 × 10 10 particles/mL) for 44 h on the growth of different bacterial species ( L. paracasei , L. acidophilus , P. acidiloctici , and Lachnospiraceae sp.). E Impact of goblet-like LS174T cells without or with NP treatment (10 6 particles/mL) for 48 h on the growth of different bacterial species ( L. paracasei, L. acidophilus, P. acidiloctici , and Lachnospiraceae sp. and Ruminococcaceae sp.). F Western blot of MUC13 inhibition by Lachnospiraceae sp.-derived EV. Data were shown as mea n ± SD ( n = 3) (* p value < 0.05). G Schematic representation summarizing the proposed mechanisms of NP-induced modulation of gut microbiota via EV. NP are taken up by Lachnospiraceae , whose EV suppress MUC13 expression in goblet cells. Concurrently, NP-modified EV from goblet cells promote the growth of Ruminococcaceae , collectively contributing to gut microbiota imbalance and potential intestinal barrier dysfunction.

Article Snippet: For Western blot, the primary antibodies for beta-actin (SC-47778, Santa Cruz, Dallas, Texas, USA), MUC13 (bs-10074R, Bioss, Boston, Massachusetts, USA), GAPDH (GTX100118, GENETEX, Texas, USA), ZO-1 (ab96587, Abcam, Bristol, UK), and OCC (ab216327, Abcam, Bristol, UK) were used to evaluated protein level.

Techniques: Bacteria, Derivative Assay, Western Blot, Inhibition, Expressing, Modification

Journal: iScience

Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance

doi: 10.1016/j.isci.2024.111139

Figure Lengend Snippet: Siglec-7 binding is enhanced by four sialyltransferases in colon cancer cell lines, and its ligand glycan is O-glycan on mucin-type proteins (A) Flow cytometry analysis of Siglec-7-Fc binding to parent DLD-1 and stably expressing cells of sialyltransferases that have been identified as Siglec-7 ligand-synthase genes ( Figure S2 A). As a result, ST3Gal1, ST6GalNAc1, ST6GalNAc3, and ST8Sia6 were identified to be capable of the induction of Siglec-7 binding. DLD-1 co-transfected with ST3Gal1 and ST6GalNAc3 genes is C3. Co-transfectant DLD-1 of ST3Gal1 and ST6GalNAc1 genes is C4. ST8Sia6 gene transfectant DLD-1 is C8. Blue-filled lines indicate negative controls, and purple lines indicate Siglec-7-Fc binding. (B and C) Effects of inhibition of N-glycosylation or O-glycosylation on the binding of Siglec-7. DLD-1 and the transfectants were treated with 20 nM kifunensine or DMSO as a negative control or 2 mM benzyl-α-GalNAc or DMSO as a negative control for 3 days, respectively. They were detached and incubated with Siglec-7-Fc (5 μg/100 μL) for 1 h and then with anti-human Fc IgG-FITC, and then analyzed using flow cytometry. (B) Kifunensine, an N-glycosylation inhibitor, showed no effect on the binding of Siglec-7. (C) Benzyl-α-GalNAc, an O-glycosylation synthesis inhibitor, led to a significant decrease in the binding of Siglec-7 in all three clones. (D) N- and O-glycan synthesis inhibition was performed on the Siglec-7-binding cell colon adenocarcinoma cell line SW837 and its effects were evaluated. (E) Biotinylated membrane proteins derived from DLD-1 and the ST transfectants were pulled down using Siglec-7-Fc or human IgG Fc only as a protein control Fc (Con-Fc) and protein A beads. (F) They were electrophoresed and transferred onto a PVDF membrane. It was used for the avidin-biotin complex detection system (ABC kit) and ECL. Specific bands were detected at 175 kDa< and 50 kDa in ST-transfectants. A specific band detected at 120 kDa in C8 was Mucin-13 (MUC13) ( Figure S2 C). (G) Immuno-blotting using anti-PODXL antibody. The upper panel indicates pull-down samples with Siglec-7-Fc or control-Fc. Lower panel indicates input lysates.

Article Snippet: The antibodies were Anti-β-Actin antibody (1:5000, Sigma), Anti-AKT (1:1000, Cell Signaling Technology), Anti- p -AKT (S473) (1:1000, Cell Signaling Technology), Anti-phospho ERK1/2 (1:1000, Cell Signaling Technology), Anti-p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology), Anti-rabbit IgG-HRP (1:1000, Cell Signaling Technology), Goat TrueBlot anti-goat Ig HRP (1:2000, ebioscience), Anti-Mouse IgG HRP-Linked Whole Ab (1:2000, GE HealthCare), Anti-MUC13 (1:1000, IMGENEX), Anti- p -Tyr (4G10) HEP conjugate (1:5000, Millipore), Anti-SHP-1 (1:1000, Millipore), Anti-Siglec-7/CD328 antibody (1:1000, R&D), Anti-PODXL (3D3) (1:1000, Santa Cruz), Anti- p -Tyr (PY20) HRP conjugate (1:2000, BD), Rat anti-PDPN (NZ-1) (1:1000, AngioBio Co), Anti-Rat IgG-HRP (1:2000, Cosmo bio), ABC standard kit Elite (5 μL/mL each, Vector) for immunoblotting, Anti-rabbit IgG Fab2 Alexa 555 (1:400, Cell Signaling Technology), Anti-mouse IgG Fab2 Alexa 647 (1:400, Cell Signaling Technology), Anti-mouse IgG Alexa 488 (1:400, Invitrogen), Phalloidin Alexa 647 (1:400, Invitrogen), DAPI (1:500, Sigma) for immunocyte chemistry.

Techniques: Binding Assay, Flow Cytometry, Stable Transfection, Expressing, Transfection, Cotransfection, Inhibition, Negative Control, Incubation, Clone Assay, Membrane, Derivative Assay, Control, Avidin-Biotin Assay

Journal: iScience

Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance

doi: 10.1016/j.isci.2024.111139

Figure Lengend Snippet:

Article Snippet: The antibodies were Anti-β-Actin antibody (1:5000, Sigma), Anti-AKT (1:1000, Cell Signaling Technology), Anti- p -AKT (S473) (1:1000, Cell Signaling Technology), Anti-phospho ERK1/2 (1:1000, Cell Signaling Technology), Anti-p44/42 MAPK (ERK1/2) (1:1000, Cell Signaling Technology), Anti-rabbit IgG-HRP (1:1000, Cell Signaling Technology), Goat TrueBlot anti-goat Ig HRP (1:2000, ebioscience), Anti-Mouse IgG HRP-Linked Whole Ab (1:2000, GE HealthCare), Anti-MUC13 (1:1000, IMGENEX), Anti- p -Tyr (4G10) HEP conjugate (1:5000, Millipore), Anti-SHP-1 (1:1000, Millipore), Anti-Siglec-7/CD328 antibody (1:1000, R&D), Anti-PODXL (3D3) (1:1000, Santa Cruz), Anti- p -Tyr (PY20) HRP conjugate (1:2000, BD), Rat anti-PDPN (NZ-1) (1:1000, AngioBio Co), Anti-Rat IgG-HRP (1:2000, Cosmo bio), ABC standard kit Elite (5 μL/mL each, Vector) for immunoblotting, Anti-rabbit IgG Fab2 Alexa 555 (1:400, Cell Signaling Technology), Anti-mouse IgG Fab2 Alexa 647 (1:400, Cell Signaling Technology), Anti-mouse IgG Alexa 488 (1:400, Invitrogen), Phalloidin Alexa 647 (1:400, Invitrogen), DAPI (1:500, Sigma) for immunocyte chemistry.

Techniques: Blocking Assay, Plasmid Preparation, Purification, Control, Staining, Recombinant, Membrane, Mutagenesis, Cytotoxicity Assay, Clone Assay, Expressing, Sequencing, Software

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Alterations in cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation

doi: 10.1152/ajpgi.00091.2024

Figure Lengend Snippet: Lack of effect of Compound-1 on MUC13 localization in MVID model mice. Immunostaining for MUC13 and ACTG1 in jejunal tissues of control, MYO5BΔIEC, and MYO5B(G519R) mice treated with Compound-1 or vehicle. In control jejunum and distal colon, MUC13 (red) is localized to the apical tips of microvilli above actin filaments (green). Both MYO5B loss-of-function mouse tissues demonstrate strong MUC13 immunostaining in cytoplasm of jejunal and colonic epithelial cells. The mislocalization of MUC13 is not remarkably altered by Compound-1 treatments in MYO5BΔIEC or MYO5B(G519R) mice. Scale bars = 20 µm. MYO5B, myosin Vb; MVID, microvillus inclusion disease.

Article Snippet: The primary antibodies against NDUFB8 (1:1,000, Abcam ab192878), HMGCS2 (1:50, Abcam ab137043), NHE3 (1:200, Novus NBP1-82574), SGLT1 (0.5 μg/mL, own) , LAMP1 (1:100, Santa Cruz sc-19992), PHH3 (1:50, Novus NBP3-08511IR, DyLight 750), Ki67 (1:100, Cell Signaling 11882S, Alexa Fluor 488), DCLK1 (1:2,000, Abcam ab202755 Alexa Fluor 647), PCNA (1:50, Santa Cruz sc-56 Alexa Fluor 647), β-catenin/CTNNB1 (12F7) (1:100, Novus NBP1-54467R, DyLight 550), villin (1:50, Santa Cruz sc-58897 Alexa Fluor 488), MUC13 (1:100, Santa Cruz, sc-390115 Alexa Fluor 546), and ACTG1 (1:100, Santa Cruz sc-65638 Alexa Fluor 546 or Alexa Fluor 790) were diluted in Dako antibody diluent with background reducing compound (S3022) and incubated on sections for 1 h at room temperature or overnight at 4°C.

Techniques: Immunostaining, Control

Journal: iScience

Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance

doi: 10.1016/j.isci.2024.111139

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-MUC13 , IMGENEX , Cat# IMG-6250A, RRID: AB_1930301.

Techniques: Blocking Assay, Plasmid Preparation, Purification, Control, Staining, Recombinant, Membrane, Mutagenesis, Cytotoxicity Assay, Cloning, Expressing, Sequencing, Software

Journal: Gut microbes

Article Title: Detecting host responses to microbial stimulation using primary epithelial organoids.

doi: 10.1080/19490976.2023.2281012

Figure Lengend Snippet: Figure 3. Development and characterization of a 2D model for primary intestinal epithelial cells. (a-c) Detection of tight junction marker, ZO1 (red), F-actin with phalloidin (green), and nuclei, DAPI (blue) (a), enterocyte marker, ALDOB (red), and nuclei, DAPI (blue) (b) and goblet cell marker, MUC13 (red), and nuclei, DAPI (blue) (c) in confluent layers of intestinal epithelial cells. Scale bar 25 µm. Insert in (a) shows a Z-section of the cell layer with clear localization of ZO1 and F-actin at the apical surface. Scale bar 10 µm. (d) Left: Primary epithelial cells seeded in 2D form a confluent layer within 7 days from seeding. Right: MA-plot based on RNA-seq analysis of 2D vs 3D cells. Y axis shows 2D vs 3D log2FC, and X shows baseline expression in TPM (transcript per million). Color shows the number of genes in each bin. Full list of GO-terms enriched in 2D and 3D cultures in Table S2. (e) Gene set enrichment analysis of the uniquely annotated genes associated with 2D or 3D cultures versus published gene signatures representing enterocyte differentiation, general differentiation in the intestine, proliferation, and stem cells. X axis show observed vs expected overlap based on randomly selected genes. (f,g) UMAP plots of single-cell RNAseq data acquired using the 10× platform. c: Colors show cells cultured in 2D and 3D. d: Colors show nine cell clusters, defined by the Leiden method. (h) Cell distribution between the different clusters from panel j. Bars

Article Snippet: The following primary antibodies were used: anti-ZO1 (61-7300; Invitrogen), anti Muc13 (HPA045163; Atlas Antibodies), and anti-AldoB (HPA073201; Atlas Antibodies) and secondary antibodies Alexa Fluor 647 polyclonal donkey anti-rabbit.

Techniques: Marker, RNA Sequencing, Expressing, Cell Culture