cyclic nucleotide 3  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 89

    Structured Review

    Millipore cyclic nucleotide 3
    Immunohistochemical staining of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and 2', <t>3'-cyclic</t> nucleotide 3'-phosphodiesterase (CNPase) in infarction and penumbra areas after I-R and I-R+EPO. (A) TTC staining of brain slices at bregma -2.30 mm after I-R. White and black arrows indicate infarction and penumbra areas. There were not seen NeuN-, GFAP-, and CNPae-immunoreactive cells in the infarction areas after I-R and I-R+EPO (B~D). The numbers of NeuN- and GFAP-immunoreactive cells in the penumbra of the I-R+EPO were not changed compared with those of I-R (B, C, I-R+EPO). However, CNPase-immunoreactive cells in the penumbra were increased under the treatment with r-Hu-EPO after I-R compared with that of I-R (D, I-R+EPO). Scale bar, 50 µm.
    Cyclic Nucleotide 3, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 8110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cyclic nucleotide 3/product/Millipore
    Average 89 stars, based on 8110 article reviews
    Price from $9.99 to $1999.99
    cyclic nucleotide 3 - by Bioz Stars, 2020-09
    89/100 stars

    Images

    1) Product Images from "Systemic injection of recombinant human erythropoietin after focal cerebral ischemia enhances oligodendroglial and endothelial progenitor cells in rat brain"

    Article Title: Systemic injection of recombinant human erythropoietin after focal cerebral ischemia enhances oligodendroglial and endothelial progenitor cells in rat brain

    Journal: Anatomy & Cell Biology

    doi: 10.5115/acb.2010.43.2.140

    Immunohistochemical staining of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in infarction and penumbra areas after I-R and I-R+EPO. (A) TTC staining of brain slices at bregma -2.30 mm after I-R. White and black arrows indicate infarction and penumbra areas. There were not seen NeuN-, GFAP-, and CNPae-immunoreactive cells in the infarction areas after I-R and I-R+EPO (B~D). The numbers of NeuN- and GFAP-immunoreactive cells in the penumbra of the I-R+EPO were not changed compared with those of I-R (B, C, I-R+EPO). However, CNPase-immunoreactive cells in the penumbra were increased under the treatment with r-Hu-EPO after I-R compared with that of I-R (D, I-R+EPO). Scale bar, 50 µm.
    Figure Legend Snippet: Immunohistochemical staining of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in infarction and penumbra areas after I-R and I-R+EPO. (A) TTC staining of brain slices at bregma -2.30 mm after I-R. White and black arrows indicate infarction and penumbra areas. There were not seen NeuN-, GFAP-, and CNPae-immunoreactive cells in the infarction areas after I-R and I-R+EPO (B~D). The numbers of NeuN- and GFAP-immunoreactive cells in the penumbra of the I-R+EPO were not changed compared with those of I-R (B, C, I-R+EPO). However, CNPase-immunoreactive cells in the penumbra were increased under the treatment with r-Hu-EPO after I-R compared with that of I-R (D, I-R+EPO). Scale bar, 50 µm.

    Techniques Used: Immunohistochemistry, Staining

    2) Product Images from "Expression of Angiopoietin 1, 2 and Their Common Receptor Tie2 in Human Gastric Carcinoma: Implication for Angiogenesis"

    Article Title: Expression of Angiopoietin 1, 2 and Their Common Receptor Tie2 in Human Gastric Carcinoma: Implication for Angiogenesis

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2006.21.2.272

    ( A-E ) In situ hybridization for Ang-1 , Ang-2 and Tie2 mRNA. ( A ) Positive Ang-2 expression in well-differentiated cancer cells (arrowheads) with no or only minimal staining on adjacent metaplastic gastric mucosa (arrows) (×100). ( B ) Moderately differentiated cancer cells show intense cytoplasmic staining for Ang-2 mRNA (×200). ( C ) Positive Ang-1 mRNA signals on cancer cells (arrowheads, ×100) as well as smooth muscle cells and endothelial cells of large vessels (arrows, Inset, ×400). ( D ) Poorly differentiated cancer cells (arrowheads) and endothelial cells (arrow) show Tie2 mRNA expression (×100). ( E ) Infiltrating poorly differentiated carcinoma cells of the muscle layer (arrowheads, ×100) show positive cytoplasmic Tie2 mRNA expression (Inset, ×400). ( F-H ) Immunohistochemistry for Ang-1, Ang-2 and Tie2 protein. ( F ) Moderately differentiated cancer cells exhibit Ang-2 protein expression (×200). ( G ) Immunoreactivity for Ang-1 on smooth muscle cells of vessels (arrows), stromal cells and cancer cells (arrowheads) (×200). ( H ) Immunohistochemistry for Tie 2. Same cancer area to Fig. E (×100) shows positive cytoplasmic expression on tumor cells (Inset, ×400).
    Figure Legend Snippet: ( A-E ) In situ hybridization for Ang-1 , Ang-2 and Tie2 mRNA. ( A ) Positive Ang-2 expression in well-differentiated cancer cells (arrowheads) with no or only minimal staining on adjacent metaplastic gastric mucosa (arrows) (×100). ( B ) Moderately differentiated cancer cells show intense cytoplasmic staining for Ang-2 mRNA (×200). ( C ) Positive Ang-1 mRNA signals on cancer cells (arrowheads, ×100) as well as smooth muscle cells and endothelial cells of large vessels (arrows, Inset, ×400). ( D ) Poorly differentiated cancer cells (arrowheads) and endothelial cells (arrow) show Tie2 mRNA expression (×100). ( E ) Infiltrating poorly differentiated carcinoma cells of the muscle layer (arrowheads, ×100) show positive cytoplasmic Tie2 mRNA expression (Inset, ×400). ( F-H ) Immunohistochemistry for Ang-1, Ang-2 and Tie2 protein. ( F ) Moderately differentiated cancer cells exhibit Ang-2 protein expression (×200). ( G ) Immunoreactivity for Ang-1 on smooth muscle cells of vessels (arrows), stromal cells and cancer cells (arrowheads) (×200). ( H ) Immunohistochemistry for Tie 2. Same cancer area to Fig. E (×100) shows positive cytoplasmic expression on tumor cells (Inset, ×400).

    Techniques Used: In Situ Hybridization, Expressing, Staining, Immunohistochemistry

    3) Product Images from "Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice"

    Article Title: Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2001.01656.x

    Immunohistochemical localization of nitrotyrosine in lung tissues from untreated (a and b) and dexamethasone (DEX)-treated mice (c) at 24 h postchallenge of P. aeruginosa It-1 strain at a dose of 2·4 × 10 5 CFU/mouse. Alveolar phagocytic cells (a and b, arrowheads) and bronchial epithelium (b, arrows) were stained with an antinitrotyrosine monoclonal antibody in the lung tissues from untreated mice, while no immunoreactivity with the same monoclonal antibody was detected in the lung tissues from DEX-treated mice (c). The lung tissue of untreated mice immediately before infection (d) was not stained with the same monoclonal antibody (d). The lung tissues of mice that received SMT as described in the Materials and methods at 24 h postinfection were not stained with antinitrotyrosine monoclonal antibody (e). Original magnification; × 200 in a, b and c, × 400 in d and e.
    Figure Legend Snippet: Immunohistochemical localization of nitrotyrosine in lung tissues from untreated (a and b) and dexamethasone (DEX)-treated mice (c) at 24 h postchallenge of P. aeruginosa It-1 strain at a dose of 2·4 × 10 5 CFU/mouse. Alveolar phagocytic cells (a and b, arrowheads) and bronchial epithelium (b, arrows) were stained with an antinitrotyrosine monoclonal antibody in the lung tissues from untreated mice, while no immunoreactivity with the same monoclonal antibody was detected in the lung tissues from DEX-treated mice (c). The lung tissue of untreated mice immediately before infection (d) was not stained with the same monoclonal antibody (d). The lung tissues of mice that received SMT as described in the Materials and methods at 24 h postinfection were not stained with antinitrotyrosine monoclonal antibody (e). Original magnification; × 200 in a, b and c, × 400 in d and e.

    Techniques Used: Immunohistochemistry, Mouse Assay, Staining, Infection

    Related Articles

    Incubation:

    Article Title: Expression of Angiopoietin 1, 2 and Their Common Receptor Tie2 in Human Gastric Carcinoma: Implication for Angiogenesis
    Article Snippet: .. Subsequently, sections were incubated with Protein Block Serum-Free (DAKO) at room temperature for 10 min and were then incubated for 2 hr at room temperature with anti-factor VIII related antigen antibody (DAKO), which stains only endothelial cells, or overnight at 4℃ with anti-angiopoietin 1, 2 or Tie2 (Chemicon International, Temecula, CA, U.S.A.) primary antibodies. .. After washing, the sections were incubated with a biotin-conjugated secondary antibody at room temperature for 30 min and finally with peroxidase conjugated streptoavidin at room temperature for 30 min. Peroxidase activity was detected with the enzyme substrate 3 amino-9-ethyl carbazole.

    Blocking Assay:

    Article Title: Expression of Angiopoietin 1, 2 and Their Common Receptor Tie2 in Human Gastric Carcinoma: Implication for Angiogenesis
    Article Snippet: .. Subsequently, sections were incubated with Protein Block Serum-Free (DAKO) at room temperature for 10 min and were then incubated for 2 hr at room temperature with anti-factor VIII related antigen antibody (DAKO), which stains only endothelial cells, or overnight at 4℃ with anti-angiopoietin 1, 2 or Tie2 (Chemicon International, Temecula, CA, U.S.A.) primary antibodies. .. After washing, the sections were incubated with a biotin-conjugated secondary antibody at room temperature for 30 min and finally with peroxidase conjugated streptoavidin at room temperature for 30 min. Peroxidase activity was detected with the enzyme substrate 3 amino-9-ethyl carbazole.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore peroxidase conjugated streptavidin
    SorLA needs complex-type N -glycosylation for shedding. (A) Schematic representation of classical N -glycan maturation in the secretory pathway. Tunicamycin blocks initiation of protein N -glycosylation, whereas MGAT1 is required for conversion of high-mannose glycans and ST3Gal sialyltransferases for capping of the glycans as indicated. Designations for monosaccharides follow the Consortium for Functional Glycomics. (B) SH-SY5Y cells expressing exogenous sorLA WT were treated with tunicamycin (1 μg/mL) for 24 h prior to biotinylation of cell surface proteins. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA. C) SH-SY5Y cells expressing exogenous sorLA WT were subjected to a 40 min [ 35 S]-labeling and chased for the indicated time points in absence or presence (+) of tunicamycin. SorLA proteins from cell lysates and medium were immunoprecipitated, and subsequent analyzed by SDS-PAGE and radio-fluorography. (D) Wild type CHO (CHO WT ) or CHO cells with targeted deletion of Mgat1 (CHO KOmgat1 ), St3gal4 and St3gal6 (CHO KOst3gal4/6 ), or St3gal3, St3gal4 , and St3gal6 (CHO KOst3gal3/4/6 ) were transfected with sorLA WT and shedding efficiency determined by WB analysis of cell lysates and conditioned medium. (E) Quantification of shed sorLA from three independent experiments as illustrated in panel D. Signals for sorLA-mat (upper band) and sorLA-im (lower band) in lysates are indicated with white and black arrowheads, respectively, while shed sorLA represented by a red arrow. The grey arrowhead indicates migration of the nascent sorLA protein in lysate with no N -glycans. (F) SH-SY5Y cells expressing exogenous sorLA WT were subjected to surface biotinylation (time 0 h) and treatment with neuraminidase (Neu) to remove sialic acids on surface located sorLA. Following cultivation at 37°C for 6 h total cell lysates were subjected to precipitation on <t>Streptavidin</t> beads (time 6 h) and analyzed by WB for detection of sorLA species. Note that surface biotinylated sorLA at time 0 h after neuraminidase treatment co-migrates with the lower migrating sorLA-im, and at time 6 h regains co-migration with the sorLA-mat upper band. To demonstrate that the surface biotinylated sorLA species detectable after 6 h indeed had regained sialic acids the streptavidin precipitate was subjected to neuraminidase again (Neu post-treatment, beads), and in agreement with this a clear shift in mobility was observed.
    Peroxidase Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peroxidase conjugated streptavidin/product/Millipore
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    peroxidase conjugated streptavidin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore hrp conjugated streptavidin
    Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with <t>streptavidin-HRP</t> and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.
    Hrp Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated streptavidin/product/Millipore
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated streptavidin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore fitc conjugated annexin v
    Effects of coptisine on cell viability and apoptosis in Hep3B cells. Hep3B cells were treated with the indicated concentrations of coptisine for 24 h. ( A ) After treated with coptisine, added the a 3-(4,5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) solution, reacted for 2 h and then detected cell viability; ( B ) The morphology of Hep3B cells by coptisine were observed by phase-contrast microscopy (magnification, ×50); ( C ) The cells were harvested and stained with annexin V-fluorescein isothiocyanate <t>(FITC)</t> and propidium iodide (PI). After staining, cells were analyzed the apoptosis by flow cytometry. Annectin V-FITC results represent early apoptosis (lower right quadrant), and late apoptosis (upper right quadrant); ( D ) Statistical analysis of <t>annexin</t> V positive cells (** p
    Fitc Conjugated Annexin V, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fitc conjugated annexin v/product/Millipore
    Average 99 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    fitc conjugated annexin v - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    SorLA needs complex-type N -glycosylation for shedding. (A) Schematic representation of classical N -glycan maturation in the secretory pathway. Tunicamycin blocks initiation of protein N -glycosylation, whereas MGAT1 is required for conversion of high-mannose glycans and ST3Gal sialyltransferases for capping of the glycans as indicated. Designations for monosaccharides follow the Consortium for Functional Glycomics. (B) SH-SY5Y cells expressing exogenous sorLA WT were treated with tunicamycin (1 μg/mL) for 24 h prior to biotinylation of cell surface proteins. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA. C) SH-SY5Y cells expressing exogenous sorLA WT were subjected to a 40 min [ 35 S]-labeling and chased for the indicated time points in absence or presence (+) of tunicamycin. SorLA proteins from cell lysates and medium were immunoprecipitated, and subsequent analyzed by SDS-PAGE and radio-fluorography. (D) Wild type CHO (CHO WT ) or CHO cells with targeted deletion of Mgat1 (CHO KOmgat1 ), St3gal4 and St3gal6 (CHO KOst3gal4/6 ), or St3gal3, St3gal4 , and St3gal6 (CHO KOst3gal3/4/6 ) were transfected with sorLA WT and shedding efficiency determined by WB analysis of cell lysates and conditioned medium. (E) Quantification of shed sorLA from three independent experiments as illustrated in panel D. Signals for sorLA-mat (upper band) and sorLA-im (lower band) in lysates are indicated with white and black arrowheads, respectively, while shed sorLA represented by a red arrow. The grey arrowhead indicates migration of the nascent sorLA protein in lysate with no N -glycans. (F) SH-SY5Y cells expressing exogenous sorLA WT were subjected to surface biotinylation (time 0 h) and treatment with neuraminidase (Neu) to remove sialic acids on surface located sorLA. Following cultivation at 37°C for 6 h total cell lysates were subjected to precipitation on Streptavidin beads (time 6 h) and analyzed by WB for detection of sorLA species. Note that surface biotinylated sorLA at time 0 h after neuraminidase treatment co-migrates with the lower migrating sorLA-im, and at time 6 h regains co-migration with the sorLA-mat upper band. To demonstrate that the surface biotinylated sorLA species detectable after 6 h indeed had regained sialic acids the streptavidin precipitate was subjected to neuraminidase again (Neu post-treatment, beads), and in agreement with this a clear shift in mobility was observed.

    Journal: bioRxiv

    Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

    doi: 10.1101/2020.07.12.199885

    Figure Lengend Snippet: SorLA needs complex-type N -glycosylation for shedding. (A) Schematic representation of classical N -glycan maturation in the secretory pathway. Tunicamycin blocks initiation of protein N -glycosylation, whereas MGAT1 is required for conversion of high-mannose glycans and ST3Gal sialyltransferases for capping of the glycans as indicated. Designations for monosaccharides follow the Consortium for Functional Glycomics. (B) SH-SY5Y cells expressing exogenous sorLA WT were treated with tunicamycin (1 μg/mL) for 24 h prior to biotinylation of cell surface proteins. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA. C) SH-SY5Y cells expressing exogenous sorLA WT were subjected to a 40 min [ 35 S]-labeling and chased for the indicated time points in absence or presence (+) of tunicamycin. SorLA proteins from cell lysates and medium were immunoprecipitated, and subsequent analyzed by SDS-PAGE and radio-fluorography. (D) Wild type CHO (CHO WT ) or CHO cells with targeted deletion of Mgat1 (CHO KOmgat1 ), St3gal4 and St3gal6 (CHO KOst3gal4/6 ), or St3gal3, St3gal4 , and St3gal6 (CHO KOst3gal3/4/6 ) were transfected with sorLA WT and shedding efficiency determined by WB analysis of cell lysates and conditioned medium. (E) Quantification of shed sorLA from three independent experiments as illustrated in panel D. Signals for sorLA-mat (upper band) and sorLA-im (lower band) in lysates are indicated with white and black arrowheads, respectively, while shed sorLA represented by a red arrow. The grey arrowhead indicates migration of the nascent sorLA protein in lysate with no N -glycans. (F) SH-SY5Y cells expressing exogenous sorLA WT were subjected to surface biotinylation (time 0 h) and treatment with neuraminidase (Neu) to remove sialic acids on surface located sorLA. Following cultivation at 37°C for 6 h total cell lysates were subjected to precipitation on Streptavidin beads (time 6 h) and analyzed by WB for detection of sorLA species. Note that surface biotinylated sorLA at time 0 h after neuraminidase treatment co-migrates with the lower migrating sorLA-im, and at time 6 h regains co-migration with the sorLA-mat upper band. To demonstrate that the surface biotinylated sorLA species detectable after 6 h indeed had regained sialic acids the streptavidin precipitate was subjected to neuraminidase again (Neu post-treatment, beads), and in agreement with this a clear shift in mobility was observed.

    Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

    Techniques: Functional Assay, Expressing, SDS Page, Western Blot, Labeling, Immunoprecipitation, Transfection, Migration

    Correlation between complex-type N -glycans, shedding, and intracellular trafficking of sorLA mutants. (A) WB analysis of lysates and medium from transfected CHO cells that express sorLA WT , sorLA FANSHY or sorLA ΔCD using a polyclonal antibody against the luminal receptor domain. (B) Cells treated with the TACE inhibitor Gm6001 (+) shows strongly reduced shed sorLA in the medium, accompanied by accumulation of the upper variant in lysates, most prominently observed for cells expressing sorLA ΔCD as these undergo much faster processing than sorLA WT . (C) SH-SY5Y cells expressing sorLA ΔCD lacking the cytosolic tail were subjected to a pulse-chase protocol in order to follow maturation and shedding over an 8 h interval. Cells and medium were harvested at the indicated time points, and sorLA ΔCD proteins were immunoprecipitated and subsequent analyzed by SDS-PAGE and radiofluorography. Quantification of mature (white) and immature (black) sorLA ΔCD from lysates is shown from three independent experiments is shown below. (D) WB analysis of lysates and medium from SH-SY5Y cells expressing sorLA ΔCD indicates loss of mature variant in the lysate caused by shedding of the high-molecular variant. (E) Cell surface proteins from SH-SY5Y cells expressing sorLA FANSHY were labeled with biotin and precipitated using Streptavidin coated beads. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA and calnexin. (F) Lysates from SH-SY5Y cells expressing sorLA FANSHY were enzymatically digested with Endo H, PNGase F, and either neuraminidase (Neu) alone or in combination with O -glycosidase. The homogenates were subsequently separated by SDS-PAGE and analyzed by WB.

    Journal: bioRxiv

    Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

    doi: 10.1101/2020.07.12.199885

    Figure Lengend Snippet: Correlation between complex-type N -glycans, shedding, and intracellular trafficking of sorLA mutants. (A) WB analysis of lysates and medium from transfected CHO cells that express sorLA WT , sorLA FANSHY or sorLA ΔCD using a polyclonal antibody against the luminal receptor domain. (B) Cells treated with the TACE inhibitor Gm6001 (+) shows strongly reduced shed sorLA in the medium, accompanied by accumulation of the upper variant in lysates, most prominently observed for cells expressing sorLA ΔCD as these undergo much faster processing than sorLA WT . (C) SH-SY5Y cells expressing sorLA ΔCD lacking the cytosolic tail were subjected to a pulse-chase protocol in order to follow maturation and shedding over an 8 h interval. Cells and medium were harvested at the indicated time points, and sorLA ΔCD proteins were immunoprecipitated and subsequent analyzed by SDS-PAGE and radiofluorography. Quantification of mature (white) and immature (black) sorLA ΔCD from lysates is shown from three independent experiments is shown below. (D) WB analysis of lysates and medium from SH-SY5Y cells expressing sorLA ΔCD indicates loss of mature variant in the lysate caused by shedding of the high-molecular variant. (E) Cell surface proteins from SH-SY5Y cells expressing sorLA FANSHY were labeled with biotin and precipitated using Streptavidin coated beads. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA and calnexin. (F) Lysates from SH-SY5Y cells expressing sorLA FANSHY were enzymatically digested with Endo H, PNGase F, and either neuraminidase (Neu) alone or in combination with O -glycosidase. The homogenates were subsequently separated by SDS-PAGE and analyzed by WB.

    Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

    Techniques: Western Blot, Transfection, Variant Assay, Expressing, Pulse Chase, Immunoprecipitation, SDS Page, Labeling

    Two sorLA variants found at the cell surface but only one is shed. (A) WB analysis of lysate and conditioned medium from SH-SY5Y cells stably transfected with sorLA WT . (B) Schematic representation of proposed N -glycosylation variants for two molecular species of sorLA (mature: arrow open, and immature: arrow filled) in lysates and one in medium (mature shed: arrow red). The high molecular weight form (upper band; sorLA-mat) carries a mixture of mature complex-type (C) and high-mannose (H) N -glycans, whereas the low molecular weight form (lower band; sorLA-im) only carries immature high-mannose N -glycans. (C) Cell surface proteins from SH-SY5Y cells expressing exogenous sorLA WT were labeled with biotin and precipitated using Streptavidin coated beads. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA and calnexin (ER marker). (D) Eluates of biotinylated cell surface proteins from SH-SY5Y and HEK293 cells expressing exogenous sorLA WT treated (+) or not (-) with neuraminidase after precipitation using Streptavidin coated beads as indicated. (E, F) SH-SY5Y cells expressing either sorLA WT or sorLA KKLN were subjected to a [ 35 S] pulse-chase protocol in order to follow the maturation over time. Cells were harvested at the indicated time points, and sorLA protein from lysate and medium was immunoprecipitated, separated by SDS-PAGE analysis, and visualized by radiofluorography. Signals in WB for lysates for sorLA-mat and sorLA-im are indicated with white and black arrowheads, and for shed sorLA in medium with a red arrowhead, respectively.

    Journal: bioRxiv

    Article Title: Endosomal trafficking is required for glycosylation and normal maturation of the Alzheimer’s-associated protein sorLA

    doi: 10.1101/2020.07.12.199885

    Figure Lengend Snippet: Two sorLA variants found at the cell surface but only one is shed. (A) WB analysis of lysate and conditioned medium from SH-SY5Y cells stably transfected with sorLA WT . (B) Schematic representation of proposed N -glycosylation variants for two molecular species of sorLA (mature: arrow open, and immature: arrow filled) in lysates and one in medium (mature shed: arrow red). The high molecular weight form (upper band; sorLA-mat) carries a mixture of mature complex-type (C) and high-mannose (H) N -glycans, whereas the low molecular weight form (lower band; sorLA-im) only carries immature high-mannose N -glycans. (C) Cell surface proteins from SH-SY5Y cells expressing exogenous sorLA WT were labeled with biotin and precipitated using Streptavidin coated beads. The biotinylated proteins together with total lysates were analyzed using SDS-PAGE and WB for sorLA and calnexin (ER marker). (D) Eluates of biotinylated cell surface proteins from SH-SY5Y and HEK293 cells expressing exogenous sorLA WT treated (+) or not (-) with neuraminidase after precipitation using Streptavidin coated beads as indicated. (E, F) SH-SY5Y cells expressing either sorLA WT or sorLA KKLN were subjected to a [ 35 S] pulse-chase protocol in order to follow the maturation over time. Cells were harvested at the indicated time points, and sorLA protein from lysate and medium was immunoprecipitated, separated by SDS-PAGE analysis, and visualized by radiofluorography. Signals in WB for lysates for sorLA-mat and sorLA-im are indicated with white and black arrowheads, and for shed sorLA in medium with a red arrowhead, respectively.

    Article Snippet: Recognition of glycosylated sorLA by lectins was carried out applying biotinylated lectins (10 μg/mL; Biotinylated Lectin Kit I and Biotinylated SNA, Vector Laboratories) and peroxidase-conjugated streptavidin (Calbiochem) diluted 1:1500.

    Techniques: Western Blot, Stable Transfection, Transfection, Molecular Weight, Expressing, Labeling, SDS Page, Marker, Pulse Chase, Immunoprecipitation

    Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with streptavidin-HRP and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.

    Journal: The Journal of Biological Chemistry

    Article Title: The endoplasmic reticulum–resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican

    doi: 10.1074/jbc.RA117.000758

    Figure Lengend Snippet: Discovery and validation of SLRP–Hsp47 interactions. A , far-Western blotting to detect potential intracellular SLRP interactants. Lysates of chondrocyte-like ATDC5 cells were run on SDS-PAGE, and the proteins were transferred onto a nitrocellulose membrane. Membrane strips were blocked with BSA and incubated with biotinylated proteins: BSA (negative control), decorin (DCN), fibromodulin (FM), or lumican (LUM). Binding was detected with streptavidin-HRP and peroxidase substrate. The arrow marks a presumed common interactant of DCN, FM, and LUM. The corresponding region was cut out from the SDS-polyacrylamide gel and analyzed by MS. B , co-immunoprecipitation assays to detect potential SLRP–Hsp47 interactions. Lysates from ATDC5 cells were used alone (−) or mixed with 1 μg of recombinant DCN, His-tagged FM, or His-tagged LUM. The lysates were incubated with decorin rabbit anti-serum ( IP : DCN ) or mouse anti-His antibodies ( IP : his-tag ) and with Protein A– or Protein G–Sepharose, respectively. Co-precipitated proteins were run on SDS-PAGE and immunoblotted for Hsp47 ( WB : HSP47 ). The capture of SLRPs was validated with the corresponding antisera ( WB : DCN , FM , LUM ). C , pulldown assays to validate SLRP–Hsp47 interactions. Magnetic beads were coated with BSA or Hsp47 (bait) and incubated with biotinylated DCN, FM, or LUM (all prey). The beads were washed, and the eluates were run on SDS-PAGE. The binding was detected by blotting with streptavidin-HRP.

    Article Snippet: Then the membrane was incubated with HRP-conjugated streptavidin diluted 1:50,000 in blocking buffer and incubated for 1 h, washed 3 × 10 min in TBST, and developed using Luminata forte (Millipore) and a CCD camera.

    Techniques: Far Western Blot, SDS Page, Incubation, Negative Control, Binding Assay, Mass Spectrometry, Immunoprecipitation, Recombinant, Western Blot, Magnetic Beads

    Effects of coptisine on cell viability and apoptosis in Hep3B cells. Hep3B cells were treated with the indicated concentrations of coptisine for 24 h. ( A ) After treated with coptisine, added the a 3-(4,5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) solution, reacted for 2 h and then detected cell viability; ( B ) The morphology of Hep3B cells by coptisine were observed by phase-contrast microscopy (magnification, ×50); ( C ) The cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). After staining, cells were analyzed the apoptosis by flow cytometry. Annectin V-FITC results represent early apoptosis (lower right quadrant), and late apoptosis (upper right quadrant); ( D ) Statistical analysis of annexin V positive cells (** p

    Journal: International Journal of Molecular Sciences

    Article Title: Induction of Apoptosis by Coptisine in Hep3B Hepatocellular Carcinoma Cells through Activation of the ROS-Mediated JNK Signaling Pathway

    doi: 10.3390/ijms21155502

    Figure Lengend Snippet: Effects of coptisine on cell viability and apoptosis in Hep3B cells. Hep3B cells were treated with the indicated concentrations of coptisine for 24 h. ( A ) After treated with coptisine, added the a 3-(4,5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) solution, reacted for 2 h and then detected cell viability; ( B ) The morphology of Hep3B cells by coptisine were observed by phase-contrast microscopy (magnification, ×50); ( C ) The cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). After staining, cells were analyzed the apoptosis by flow cytometry. Annectin V-FITC results represent early apoptosis (lower right quadrant), and late apoptosis (upper right quadrant); ( D ) Statistical analysis of annexin V positive cells (** p

    Article Snippet: The collected cells were re-suspended in binding buffer, and then stained with PI solution and FITC-conjugated annexin V, for 15 min, in dark, as previously described [ ].

    Techniques: MTT Assay, Microscopy, Staining, Flow Cytometry