Journal: International journal of molecular sciences

Article Title: Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

doi: 10.3390/ijms232012626

Figure Lengend Snippet: Figure 4. Dependence of Au/CD44/anti-CD44 complex dissociation and surface regeneration efficiency on the nature of the regeneration solution.

Article Snippet: Recombinant human CD44 biomarker (career free, His tag C-Termus), monoclonal mouse IgG clone # 2C5 (anti-CD44) and recombinant human growth hormone from E. coli (hGH) were obtained from R&D Systems (Abingdon, UK).

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Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: Colocalization of CD44HSPG and MMP-7 in cell lines. (A) (Top panel) Phorbolester-treated neuroblastoma SH-SY5Y cells display colocalization of MMP-7 and CD44HSPG on the cell surface and along neurite extensions (inset). (Bottom panel) CHO cells cotransfected with cDNAs encoding human CD44v3-10 and MMP-7 display colocalization of the two molecules in cell protrusions. (B) Recombinant purified MMP-7 binds weakly to Ltk− [L-M(TK−)] cells, but strongly to Ltk− cells transfected with CD44v3-10. Binding is abrogated by washing the transfectants with 0.2 mg/mL heparin.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Recombinant, Purification, Transfection, Binding Assay

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: Reconstitution of the CD44HSPG/MMP 7/HB-EGF/ErB4 complex in Namalwa cells. (A) Confocal microscopy of Namalwa CD44 transfectants incubated with anti-CD44 (red fluorescence) and anti-MMP-7 (green fluorescence) mAb. Namalwa transfectants expressing CD44v3-10 (top) and CD44H (bottom) are shown. (B) Coimmunoprecipitation of CD44v3 and MMP-7. Indicated Namalwa transfectants were incubated with anti-MMP-7 C-M7 antibody, washed extensively, and lysed. Lysates were incubated with protein A sepharose beads and the immunoprecipitates were subjected to SDS-PAGE, transferred onto Hybond-C nitrocellulose membranes and immunoblotted with anti-CD44 mAb. (C) Transferrin zymogram of heparin eluates from anti-CD44 antibody immunoprecipitates of Namalwa CD44v3.8-10 and CD44v3-10 transfectant lysates (left). Transferrin zymogram of chondroitin lyase (Ch)− and heparanase (Hep)-treated anti-CD44 mAb immunoprecipitates from CD44v3-10 and CD44v7-10 Namalwa transfectants (right). C1 and C2 are control lanes containing protein A beads only (C1) and heparinase, chondroitinase, and digestion buffer in the absence of substrate (C2). MMP-3 (M3) and pro- and active-recombinant MMP-7 (M7) are used as marker controls. Migration of pro- and active MMP-7 are indicated on the left along with the molecular weight markers.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Confocal Microscopy, Incubation, Fluorescence, Expressing, SDS Page, Transfection, Recombinant, Marker, Migration, Molecular Weight

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: HB-EGF association with and processing by MMP 7. (A) (Top panel) Localization of HB-EGF on the surface of CD44v3 isoform expressing Namalwa cells, as assessed by neutralizing anti-HB-EGF antibody reactivity. (Bottom panel) Colocalization of proHB-EGF (C-18 antibody) and MMP-7 (RM7-C antibody) in Namalwa cells transfected with CD44v3-10, as assessed by confocal microscopy (magnification, 63×). (B) Coimmunoprecipitation of HB-EGF and MMP-7. MMP-7 was immunoprecipitated from Namalwa cells expressing the indicated CD44 isoforms with the indicated anti-MMP-7 antibody. The immunoprecipitates were subjected to SDS-PAGE, transferred onto nylon filters, and immunoblotted with the indicated anti-HB-EGF mAb. Lanes indicated pep C-18 and Mock C, respectively denote immunoprecipitates in the presence of immunogenic peptide and immunoprecipitates derived from vector only transfectants. (C) Proteolytic cleavage of pro-HB-EGF by MMP-7. Namalwa transfectant-derived anti-CD44 mAb immunoprecipitates were washed with a buffer containing EDTA to remove the coimmunoprecipitated MMP-7 and any other possibly associated MMP activity. The immunoprecipitates were then incubated with recombinant active MMP-7 in the presence or absence of the synthetic inhibitor SC44463 and subjected to SDS-PAGE, transfer to nitrocellulose membranes, and immunoblotting with a combination of neutralizing and cytoplasmic domain anti-HB-EGF mAb. Molecular weight markers are indicated.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Expressing, Transfection, Confocal Microscopy, Immunoprecipitation, SDS Page, Derivative Assay, Plasmid Preparation, Activity Assay, Incubation, Recombinant, Western Blot, Molecular Weight

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: . ErbB4 complexes with CD44 but is phosphorylated preferentially when associated with CD44v3. (A) Confocal microscopy shows colocalization of ErbB4 (red fluorescence) and CD44 (green fluorescence) in Namalwa CD44 transfectants. (B) (Top panel) Western blot analysis of anti-CD44 mAb immunoprecipitates from Namalwa transfectant lysates using anti-phosphotyrosine mAb pY20. (Bottom panel) Western blot analysis of anti-CD44 mAb immunoprecipitates in B immunoblotted with anti-c-ErbB4 mAb HFR-1. (C) HB-EGF neutralizing Ab (100 ng/mL), proMMP-7E219Q mutant (300 ng/mL), TIMP-3 (100 ng/mL), and MMP inhibitors SC44463 and BB94 (50 ng/mL) can reduce the level of tyrosine phosphorylated ErbB-4 in CD44V3-10 transfected Namalwa cells. (Top panel) Anti-tyrosine Ab pY20 blot. (Bottom panel) Anti-C-erbB-4 Ab-4 (HFR-1) blot. (D) Pro-MMP-7E219Q binds to the surface of Namalwa CD44HSPG and promotes apoptosis in serum-free medium. Namalwa transfectants expressing CD44v3-10 were incubated in serum free medium in the presence or absence of 100 ng/mL of recombinant proMMP-7E219Q alone or proMMP-7E219Q and recombinant active HB-EGF (50 ng/mL) as indicated. The cells were then tested for cell surface-bound MMP 7E219Q, using rat N-terminal propeptide MMP-7-specific antibody RM7-N (red fluorescence), and apoptosis, as assessed by TUNEL staining (green fluorescence). The RM7-N antibody did not react with CD44v3-10 Namalwa transfectants in the absence of exogenous rat proMMP 7E219Q (left). RM7-N stained CD44v3-10 Namalwa transfectants incubated with pro-MMP 7E219Q, but not CD44 transfectants lacking HSPG (data not shown). The majority of MMP-7E219Q bound cells tested TUNEL positive. Recombinant active HB-EGF (50 ng/mL) bound to the surface of transfectants preincubated with proMMP 7E219Q, as assessed by staining with neutralizing anti-HB-EGF antibody (right) and could rescue the majority of the cells from MMP 7E219Q binding-induced apoptosis.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Confocal Microscopy, Fluorescence, Western Blot, Transfection, Mutagenesis, Expressing, Incubation, Recombinant, TUNEL Assay, Staining, Binding Assay

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: CD44 regulates postpartum uterus remodeling. (A). Gross anatomy (a) and histology (b–g) of 36 h postpartum uteri from wild-type (+/+) and CD44 deficient (−/−) DBA/l mice. Note the comparable diameter of the cervix (a). Low power (8×) magnification of uterine cross-sections shows a preserved endometrial and myometrial structure in wild-type postpartum uterus (b) that contrasts with disorganized architecture of the CD44−/− counterpart (c), characterized by the loss of myometrial mass. Higher magnification (40×) shows predominantly round nuclei in the epithelial cells of wild-type uterus (d) compared to elongated, hyperchromatic nuclei in the more columnar epithelial cells of CD44−/− uterus (e). Smooth muscle layers are preserved in wild-type uterus (f), but appear disorganized and condensed, with hyperchromatic nuclei in CD44−/− uterus (g). (B) CD44HSPG-dependent localization of MMP-7 to the apical surface of uterine epithelium. (Top panels) 36 h postpartum uteri from wild-type and CD44−/− mice were stained with anti-CD44v3 mAb (red fluorescence) and anti-MMP-7 mAb (green fluorescence). (Bottom panels) Comparison of the localization of anti-MMP-7 antibody staining to that of anti-basement membrane proteoglycan perlecan antibody staining in 36 h postpartum epithelium of wild-type and CD44−/− mice. White and red arrows indicate the apical and basal epithelial surface, respectively. (C) MMP-7 and HB-EGF localization in wild-type and CD44−/− uterine epithelium. Thirty-six h postpartum uteri from wild-type and CD44−/− mice were stained with anti-MMP-7 antibody (red fluorescence) and anti-HB-EGF neutralizing antibody that recognizes the mature form only (HB-EGF), or the C-18 antibody against a peptide in the cytoplasmic tail of pro-HB-EGF (proHB-EGF, green fluorescence). White and red arrows denote the apical and basal epithelial surface, respectively. (D) MMP-7 and HB-EGF localization in wild-type and CD44−/− postpartum uterine smooth muscle. Staining was performed as in C and shows colocalization of MMP-7 and mature HB-EGF in wild-type smooth muscle cells (top). In CD44−/− postpartum uteri, MMP-7 is redistributed to the cell periphery, MMP-7 and pro-HB-EGF show only occasional colocalization (middle), and mature HB-EGF is barely detectable (bottom). (E) Colocalization of ErbB4 and phosphotyrosine in postpartum uterine epithelium. Anti-ErbB4 antibody (red fluorescence) and anti-phosphotyrosine pY20 mAb (green fluorescence) predominantly stain the apical epithelial surface in wild-type postpartum uterus (top). In CD44−/− postpartum uterus, ErbB4 distribution is unchanged, whereas apical epithelial tyrosine phosphorylation is abrogated, and only intracellular phosphorylation remains detectable. White and red arrows indicate the apical and basal epithelial surface, respectively (F) TUNEL (green fluorescence) and anti-CD44 mAb staining (red fluorescence) in wild-type and CD44−/− 48 h postpartum uteri. Wild-type (top) and CD44−/− (second panel) epithelial, and wild-type (third panel), and CD44−/− smooth muscle (bottom) staining is shown, indicating massive apoptotic cell death in both cell types in the absence of CD44.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Staining, Fluorescence, Comparison, Membrane, TUNEL Assay