• Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    Name:
    Adenosine 5 Triphosphate disodium salt
    Description:
    Adenosine 5 Triphosphate disodium salt is a p2 purinergic P2X and P2Y agonist that increases Ca2 activated K channel activity It has been found to induce a release of prostoglandin D2 and histamine from rat peritoneal mast cells in a dose dependent manner
    Catalog Number:
    SC-202040
    Price:
    None
    Category:
    Chemicals Protein Interacting Inhibitors Activators Substrates Protein Activators P2X Activators Adenosine 5 Triphosphate disodium salt
    Applications:
    Adenosine 5′-Triphosphate, disodium salt is a p2 purinergicagonist
    Purity:
    ≥98%
    Molecular Weight:
    551.14
    Buy from Supplier


    Structured Review

    Santa Cruz Biotechnology atp
    Graphical summary of results. Upon agonist-induced platelet activation, <t>ATP</t> and <t>ADP</t> are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein
    Adenosine 5 Triphosphate disodium salt is a p2 purinergic P2X and P2Y agonist that increases Ca2 activated K channel activity It has been found to induce a release of prostoglandin D2 and histamine from rat peritoneal mast cells in a dose dependent manner
    https://www.bioz.com/result/atp/product/Santa Cruz Biotechnology
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2021-07
    91/100 stars

    Images

    1) Product Images from "Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine"

    Article Title: Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0936-8

    Graphical summary of results. Upon agonist-induced platelet activation, ATP and ADP are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein
    Figure Legend Snippet: Graphical summary of results. Upon agonist-induced platelet activation, ATP and ADP are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein

    Techniques Used: Activation Assay, Activity Assay, ALP Assay

    2) Product Images from "Ginsenoside Rg1 alleviates acute liver injury through the induction of autophagy and suppressing NF-κB/NLRP3 inflammasome signaling pathway"

    Article Title: Ginsenoside Rg1 alleviates acute liver injury through the induction of autophagy and suppressing NF-κB/NLRP3 inflammasome signaling pathway

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.50919

    The roles of autophagy and NLRP3 inflammasome in mice with CCl 4 -induced acute liver injury. (A) Serum AST, ALT, TNF-α, IL-1β, IL-6 were measured in control group, RPA group and 3-MA group 24 h after the CCl 4 injection. (B) Serum AST, ALT, TNF-α, IL-1β, IL-6 were measured in control group, MCC950 group and ATP group 24 h after the CCl 4 injection (n=3). *P
    Figure Legend Snippet: The roles of autophagy and NLRP3 inflammasome in mice with CCl 4 -induced acute liver injury. (A) Serum AST, ALT, TNF-α, IL-1β, IL-6 were measured in control group, RPA group and 3-MA group 24 h after the CCl 4 injection. (B) Serum AST, ALT, TNF-α, IL-1β, IL-6 were measured in control group, MCC950 group and ATP group 24 h after the CCl 4 injection (n=3). *P

    Techniques Used: Mouse Assay, AST Assay, Recombinase Polymerase Amplification, Injection

    3) Product Images from "Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine"

    Article Title: Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0936-8

    Graphical summary of results. Upon agonist-induced platelet activation, ATP and ADP are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein
    Figure Legend Snippet: Graphical summary of results. Upon agonist-induced platelet activation, ATP and ADP are released. These are converted to AMP by platelet CD39 activity. AMP is converted to adenosine by MSC-expressed CD73 and to a low extent by alkaline phosphatase. Adenosine signals vial A2AR and other P1 receptors to raise cAMP levels and to induce VASP phosphorylation. This reduces further platelet activation. Used inhibitors indicated in red. ADA adenosine deaminase, ADP adenosine diphosphate, ALP alkaline phosphatase, AMP adenosine monophosphate, ATP adenosine triphosphate, TRAP thrombin receptor activator for peptide, VASP vasodilator-stimulated phosphoprotein

    Techniques Used: Activation Assay, Activity Assay, ALP Assay

    Related Articles

    other:

    Article Title: P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line
    Article Snippet: Adenosine 5′-triphosphate (ATP) and benzoyl adenosine 5′-triphosphate (BzATP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A); A740003 was obtained from Tocris (Ellisville, MO, USA); suramin was obtained from RBI (Natick, Mass., USA); reactive blue 2 (RB2), ethidium bromide was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) and anti-P2X7 receptor antibody was obtained from Alamone labs (Jerusalem, Israel).

    Incubation:

    Article Title: Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine
    Article Snippet: Detection of ectonucleotidase activity Ectonucleotidase activity was measured in the cells as described previously [ ]. .. Briefly, cells were seeded at 10,000 cells/cm2 in 24-well plates and then incubated for 24 h. ATP, ADP and AMP (1 mM; Santa Cruz) were then added in phosphate-free buffer and incubated for either 1 h (ATP and ADP) or 30 min (AMP). .. Supernatant was harvested for protein quantification (BCA assay; Thermo Fisher, Waltham, MA, USA), inorganic phosphate quantification (malachite green assay kit, according to the manufacturer’s instructions; Sigma Aldrich) or adenosine detection.

    Article Title: Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates
    Article Snippet: To measure ATP hydrolysis, we used the ADP-Glo™ Kinase Assay (Promega) according to the manufacturer’s instructions. .. Briefly, 5 μl of renatured proteins were mixed in a white 96-well plate (Santa Cruz Biotechnology) with ATP (100 uM final concentration) and 0.1 μl RNase A/T1 mixture or vehicle (50% Glycerol in 20 mM Tris-HCl pH 7.5), all diluted in 1X PBS, 5 mM MgCl2, 2 mM DTT, in a total volume of 15 μl and incubated at room temperature for 1.5 hour. .. Non-hydrolysed ATP was removed by the addition of 15 μl of ADP-Glo reagent followed by incubation for 1 hour at room temperature.

    Concentration Assay:

    Article Title: Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates
    Article Snippet: To measure ATP hydrolysis, we used the ADP-Glo™ Kinase Assay (Promega) according to the manufacturer’s instructions. .. Briefly, 5 μl of renatured proteins were mixed in a white 96-well plate (Santa Cruz Biotechnology) with ATP (100 uM final concentration) and 0.1 μl RNase A/T1 mixture or vehicle (50% Glycerol in 20 mM Tris-HCl pH 7.5), all diluted in 1X PBS, 5 mM MgCl2, 2 mM DTT, in a total volume of 15 μl and incubated at room temperature for 1.5 hour. .. Non-hydrolysed ATP was removed by the addition of 15 μl of ADP-Glo reagent followed by incubation for 1 hour at room temperature.

    Article Title: CDKL3 promotes osteosarcoma progression by activating Akt/PKB
    Article Snippet: Methyl thiazolyl tetrazolium (MTT) assay for cell proliferation Cells transducted with lentivirus expressing indicated shRNAs were plated on 96-well plates at a density of 3,000 cells per well in triplicates and incubated for 1–5 d. MTT (thiazolyl blue tetrazolium, from Sigma-Aldrich) was added into each well for a final concentration of 0.5 mg/ml, and the plates were incubated in 37°C for additional 4 h. After the incubation, all the medium was removed and 100 μl of DMSO was added to each well. .. At the condition of adding ATP (from Santa Cruz Biotech, final concentration in reaction: 0.2 mM) or distilled water, the reactions were performed at 30° for 30 min. Then, the reactions were stopped immediately by adding 3× SDS-containing Laemmli buffer at due time. .. Western blot and Co-IP The antibodies were purchased from the following companies: Cell Signaling Technology: LC3, Pan-Akt (Rb and Mo), Akt pT308, Akt pT450, GAPDH, p70S6K, p70-S6K pT389, mTOR, GSK3β, GSK3β pS9, 4E-BP1, p4E-BP1, Atg12, Beclin-1, HA, myc, GST, and IgG; Invitrogen: CDKL3 (NKIAMRE) and Akt pS473; ImmunoWay: mTOR pS2448 and mTOR pS2481; ProteinTech: FoxO1 and FoxO3a; and Santa Cruz Biotech: β-actin and protein G agarose.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Santa Cruz Biotechnology goat anti hlamp 2
    Development of <t>anti-hLAMP-2</t> ELISA. (A) Comparison of ELISA plates coated with 0.5 and 5.0 µg/ml of recombinant hLAMP-2/GST fusion protein (FP). Plates coated with 5.0 µg/ml gave much better separation between positive and negative sera. (B) ELISA plates coated with hLAMP-2/GST FP were stored at 4°C and tested after different time points. Serial dilutions of a standard positive control serum gave similar results using plates stored for 1–33 days, but there was a considerable increase in background binding after 41 days. (C) Comparison of ELISA plates coated with two different batches of hLAMP-2/GST FP demonstrates comparable binding of serial dilutions of a standard positive control serum. (D) The standard curve derived from the results of assaying serial dilutions of the standard control serum was used to calculate anti-hLAMP-2 antibody concentrations with 100 U equating to OD of the 1:100 dilution of the positive control.
    Goat Anti Hlamp 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti hlamp 2/product/Santa Cruz Biotechnology
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti hlamp 2 - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology h 89 dihydrochloride
    PKA but not PKCε plays a role in the expression of Type II priming induced by repeated exposure to CPA A. Male rats that were treated with repeated intradermal administration of CPA (1 µg, hourly, x4) on the dorsum of the hind paw received, 4 days later, an injection of PGE 2 (100 ng) at the same site, in the presence of vehicle (control, black bars) or the PKCε inhibitor PKCε V1-2 (1 µg, white bars), administered 10 min before. Mechanical nociceptive threshold was evaluated 30 min and 4 h after PGE 2 . We observed no difference between the groups in the prolongation of the PGE 2 -induced hyperalgesia ( F 1,10 = 0.27; p = 0.6167, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the prolongation of PGE 2 -induced hyperalgesia in Type II priming produced by CPA is not dependent on PKCε; B ( Left panel ). Rats were treated with intradermal injection of vehicle (control, black bars) or <t>H-89</t> (1 µg, white bars) on the dorsum of the hind paw. 10 min later, 4 injections of CPA (1 µg, hourly) were performed in both groups of rats. We observed, in the group pretreated with H-89, significant attenuation of the mechanical hyperalgesia induced by the 4 th injection of CPA, when compared with the control group ( F 1,10 = 22.76; *** p
    H 89 Dihydrochloride, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h 89 dihydrochloride/product/Santa Cruz Biotechnology
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h 89 dihydrochloride - by Bioz Stars, 2021-07
    93/100 stars
      Buy from Supplier

    99
    Santa Cruz Biotechnology mouse igg1 anti na k atpase α1
    Expression of cultured P3 hCECs. Representative sets of photomicrographs showing expression of Na + K + <t>/ATPase</t> and ZO-1 by immunocytochemistry: Immunostaining of Na + K + /ATPase in A : M2 and B : M4. Immunostaining of ZO-1 in C : M2 and D : M4. Control staining E : Isotype matched <t>IgG</t> 1 negative control. ( n = 6; Scale bars = 50 µm).
    Mouse Igg1 Anti Na K Atpase α1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse igg1 anti na k atpase α1/product/Santa Cruz Biotechnology
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse igg1 anti na k atpase α1 - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology poly adp ribose polymerase
    Activation of caspase-3 by 7-ketocholesterol is reduced in <t>CB2</t> deficient macrophages. Resident peritoneal macrophages, isolated and pooled from CB2 +/+ and CB2 −/− mice, were cultured for 16 h in the presence or absence of 10 μg/ml 7KC. A: Caspase-3 activity was determined as described in Materials and Methods, and the results are present as the mean fold induction ± SD for three independent experiments. B: Total cell lysates prepared from CB2 +/+ and CB2 −/− MPMs treated with and without 7KC for 16 h were subjected to immunoblotting using antibodies specific for procaspase-3, cleaved caspase-3 and <t>poly-ADP</t> ribose polymerase (PARP). The blots were striped and probed with an Hsc70 antibody to control for equal loading of the gels. The blots shown are representative of three independent experiments. C: The relative amounts of procaspase-3, cleaved caspase-3, full length PARP and cleaved PARP detected by the immunoblots from the three replicate experiments were quantified by densitometric analysis, normalized to Hsc70 levels, and expressed as the mean ratio of cleaved caspase-3 / procaspase-3 ± SD (C) and as the mean ratio of cleaved PARP / total PARP ± SD, respectively (D). * P
    Poly Adp Ribose Polymerase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/poly adp ribose polymerase/product/Santa Cruz Biotechnology
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    poly adp ribose polymerase - by Bioz Stars, 2021-07
    93/100 stars
      Buy from Supplier

    Image Search Results


    Development of anti-hLAMP-2 ELISA. (A) Comparison of ELISA plates coated with 0.5 and 5.0 µg/ml of recombinant hLAMP-2/GST fusion protein (FP). Plates coated with 5.0 µg/ml gave much better separation between positive and negative sera. (B) ELISA plates coated with hLAMP-2/GST FP were stored at 4°C and tested after different time points. Serial dilutions of a standard positive control serum gave similar results using plates stored for 1–33 days, but there was a considerable increase in background binding after 41 days. (C) Comparison of ELISA plates coated with two different batches of hLAMP-2/GST FP demonstrates comparable binding of serial dilutions of a standard positive control serum. (D) The standard curve derived from the results of assaying serial dilutions of the standard control serum was used to calculate anti-hLAMP-2 antibody concentrations with 100 U equating to OD of the 1:100 dilution of the positive control.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

    doi: 10.1681/ASN.2011090920

    Figure Lengend Snippet: Development of anti-hLAMP-2 ELISA. (A) Comparison of ELISA plates coated with 0.5 and 5.0 µg/ml of recombinant hLAMP-2/GST fusion protein (FP). Plates coated with 5.0 µg/ml gave much better separation between positive and negative sera. (B) ELISA plates coated with hLAMP-2/GST FP were stored at 4°C and tested after different time points. Serial dilutions of a standard positive control serum gave similar results using plates stored for 1–33 days, but there was a considerable increase in background binding after 41 days. (C) Comparison of ELISA plates coated with two different batches of hLAMP-2/GST FP demonstrates comparable binding of serial dilutions of a standard positive control serum. (D) The standard curve derived from the results of assaying serial dilutions of the standard control serum was used to calculate anti-hLAMP-2 antibody concentrations with 100 U equating to OD of the 1:100 dilution of the positive control.

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-hLAMP-2, clone H4B4 (Developmental Studies Hybridoma Bank, University of Iowa); polyclonal rabbit ( ; Abnova, Taipei, Taiwan); goat anti-hLAMP-2 (sc-8101; Santa Cruz biotechnology, Santa Cruz, CA); and rabbit anti-rLAMP-2 (Zymed Laboratories, San Francisco, CA).

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Binding Assay, Derivative Assay

    Antibodies to hLAMP-2 in patients with AAV. (A) ELISA results from patients presenting with active piFNGN/AAV from Vienna ( n =14), Groningen ( n =44), and Cambridge ( n =33). The upper limit of normal is 29 and the shaded area between 22 and 29 indicates borderline. Sera confirmed to have anti-hLAMP-2 antibodies by the other two assays are in red, whereas those without anti-hLAMP-2 antibodies are shown in green. Both ELISA and IIF assays were negative in sera in green with positive ELISA. (B) None of the 30 controls from Vienna with various types of renal disease were judged to have anti-hLAMP-2 antibodies, although 4 patients had isolated positive ELISA with negative Western blots and IIF assays. Two of the 21 SLE patients (9.5%) had anti-hLAMP-2 antibodies and an additional 2 patients had positive ELISA not confirmed by the other two assays. (C) Proportion of patients the Vienna, Groningen, and Cambridge cohorts with active piFNGN/AAV with antibodies to LAMP-2. Results are shown separately for untreated patients at presentation, patients presenting after immunosuppressive treatment had been started, and sera taken during relapse. The overall frequency of antibodies to LAMP-2 at presentation was 82% and was higher in untreated patients than those already on treatment. This difference was significant for the whole group ( P =0.0071, Fisher's exact text) and for the Groningen cohort ( P =0.0236, Fisher’s exact test).

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

    doi: 10.1681/ASN.2011090920

    Figure Lengend Snippet: Antibodies to hLAMP-2 in patients with AAV. (A) ELISA results from patients presenting with active piFNGN/AAV from Vienna ( n =14), Groningen ( n =44), and Cambridge ( n =33). The upper limit of normal is 29 and the shaded area between 22 and 29 indicates borderline. Sera confirmed to have anti-hLAMP-2 antibodies by the other two assays are in red, whereas those without anti-hLAMP-2 antibodies are shown in green. Both ELISA and IIF assays were negative in sera in green with positive ELISA. (B) None of the 30 controls from Vienna with various types of renal disease were judged to have anti-hLAMP-2 antibodies, although 4 patients had isolated positive ELISA with negative Western blots and IIF assays. Two of the 21 SLE patients (9.5%) had anti-hLAMP-2 antibodies and an additional 2 patients had positive ELISA not confirmed by the other two assays. (C) Proportion of patients the Vienna, Groningen, and Cambridge cohorts with active piFNGN/AAV with antibodies to LAMP-2. Results are shown separately for untreated patients at presentation, patients presenting after immunosuppressive treatment had been started, and sera taken during relapse. The overall frequency of antibodies to LAMP-2 at presentation was 82% and was higher in untreated patients than those already on treatment. This difference was significant for the whole group ( P =0.0071, Fisher's exact text) and for the Groningen cohort ( P =0.0236, Fisher’s exact test).

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-hLAMP-2, clone H4B4 (Developmental Studies Hybridoma Bank, University of Iowa); polyclonal rabbit ( ; Abnova, Taipei, Taiwan); goat anti-hLAMP-2 (sc-8101; Santa Cruz biotechnology, Santa Cruz, CA); and rabbit anti-rLAMP-2 (Zymed Laboratories, San Francisco, CA).

    Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Western Blot

    Indirect immunofluorescence on ldlD cells stably expressing hLAMP-2 on the cell surface (ldlD/hLAMP-2H). (A) ldlD cells were transfected with full-length hLAMP-2 cDNA with a tyrosine to histidine mutation in the cytoplasmic lysosomal retrieval signal. Stable transfectants were sorted by flow cytometry for uniform expression of the transgene and stained with monoclonal (CD3) and polyclonal (932B) antibodies to hLAMP-2 generated by M. Fukuda as well as commercially available monoclonal (H4B4) and polyclonal anti-LAMP-2 antibodies (SCN17 and Abnova). All of the antibodies bound to hLAMP-2 on the surface of nonpermeabilized transfected cells (np). The commercial anti-hLAMP-2 cross-reacted with hamster LAMP-2 in transfected and untransfected cells especially after permeabilization (p). CD3 and 932B were the only antibodies that did not cross-react with hamster LAMP-2. A polyclonal antibody to rat LAMP-2 (Zymed Laboratories) and secondary antibody alone fail to detect either human or hamster LAMP-2. (B) FACS analysis of ldlD/hLAMP-2H cells and control ldlD cells stained with H4B4, a mAb to hLAMP-2 that cross-reacts with hamster LAMP-2. Surface expression of hLAMP-2 is detected only in transfected cells, whereas intracellular staining of native hamster LAMP-2 is apparent after permeabilization with saponin. (C) The sensitivity of the IIF assay for antibodies to hLAMP-2 assessed from doubling dilutions (1:50 to 1:800) of the standard positive control (Stand Co) compared with binding of monoclonal anti-hLAMP-2 antibody (H4B4; pos Co) and negative control serum (neg Co). IgG binding to ldlD/hLAMP-2H cells decreases progressively in titers > 1:100 and becomes negative at 1:400, which is 10-fold higher than the standard assay dilution of 1:40.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

    doi: 10.1681/ASN.2011090920

    Figure Lengend Snippet: Indirect immunofluorescence on ldlD cells stably expressing hLAMP-2 on the cell surface (ldlD/hLAMP-2H). (A) ldlD cells were transfected with full-length hLAMP-2 cDNA with a tyrosine to histidine mutation in the cytoplasmic lysosomal retrieval signal. Stable transfectants were sorted by flow cytometry for uniform expression of the transgene and stained with monoclonal (CD3) and polyclonal (932B) antibodies to hLAMP-2 generated by M. Fukuda as well as commercially available monoclonal (H4B4) and polyclonal anti-LAMP-2 antibodies (SCN17 and Abnova). All of the antibodies bound to hLAMP-2 on the surface of nonpermeabilized transfected cells (np). The commercial anti-hLAMP-2 cross-reacted with hamster LAMP-2 in transfected and untransfected cells especially after permeabilization (p). CD3 and 932B were the only antibodies that did not cross-react with hamster LAMP-2. A polyclonal antibody to rat LAMP-2 (Zymed Laboratories) and secondary antibody alone fail to detect either human or hamster LAMP-2. (B) FACS analysis of ldlD/hLAMP-2H cells and control ldlD cells stained with H4B4, a mAb to hLAMP-2 that cross-reacts with hamster LAMP-2. Surface expression of hLAMP-2 is detected only in transfected cells, whereas intracellular staining of native hamster LAMP-2 is apparent after permeabilization with saponin. (C) The sensitivity of the IIF assay for antibodies to hLAMP-2 assessed from doubling dilutions (1:50 to 1:800) of the standard positive control (Stand Co) compared with binding of monoclonal anti-hLAMP-2 antibody (H4B4; pos Co) and negative control serum (neg Co). IgG binding to ldlD/hLAMP-2H cells decreases progressively in titers > 1:100 and becomes negative at 1:400, which is 10-fold higher than the standard assay dilution of 1:40.

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-hLAMP-2, clone H4B4 (Developmental Studies Hybridoma Bank, University of Iowa); polyclonal rabbit ( ; Abnova, Taipei, Taiwan); goat anti-hLAMP-2 (sc-8101; Santa Cruz biotechnology, Santa Cruz, CA); and rabbit anti-rLAMP-2 (Zymed Laboratories, San Francisco, CA).

    Techniques: Immunofluorescence, Stable Transfection, Expressing, Transfection, Mutagenesis, Flow Cytometry, Cytometry, Staining, Generated, FACS, Positive Control, Binding Assay, Negative Control

    Evaluation of assays for antibodies to LAMP-2. (A) ELISA results from a panel of sera from 78 healthy controls were used to derive the mean ± SD for this group and the 95% confidence limit of the upper limit of normal for the assay, which was established at 29 U. Six controls had positive ELISA with negative Western blots and IIF assays. (B) ELISA results of the data shown in A. The upper limit of normal in the assay is 29 U. Sera confirmed to have anti-hLAMP-2 antibodies by the other two assays are in red, whereas those in which positive ELISA was not confirmed because ELISA and IIF assays were negative are in green. (C) Measurement of antibodies to hLAMP-2 by ELISA and Western blot using hLAMP-2/GST FP and IIF on ldlD/hLAMP-2H cells was compared using a panel of 41 sera selected to cover a range of positive and negative values. The panel consisted of 16 sera from patients with AAV (11 with active disease and 5 in remission), 15 controls with other renal diseases, and 10 healthy controls. The assays were graded positive (red), low positive (Western and IIF), borderline (ELISA) (orange), or negative (green). The figure illustrates the strong concordance among results from the three assays. Sera were considered to have antibodies to LAMP-2 when ≥2 assays were positive. Abbreviations: TX, renal transplant; MGN, membranous nephropathy; IgA GN, IgA nephropathy; MPGN, membranoproliferative GN; TIN, tubulointerstitial nephritis.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

    doi: 10.1681/ASN.2011090920

    Figure Lengend Snippet: Evaluation of assays for antibodies to LAMP-2. (A) ELISA results from a panel of sera from 78 healthy controls were used to derive the mean ± SD for this group and the 95% confidence limit of the upper limit of normal for the assay, which was established at 29 U. Six controls had positive ELISA with negative Western blots and IIF assays. (B) ELISA results of the data shown in A. The upper limit of normal in the assay is 29 U. Sera confirmed to have anti-hLAMP-2 antibodies by the other two assays are in red, whereas those in which positive ELISA was not confirmed because ELISA and IIF assays were negative are in green. (C) Measurement of antibodies to hLAMP-2 by ELISA and Western blot using hLAMP-2/GST FP and IIF on ldlD/hLAMP-2H cells was compared using a panel of 41 sera selected to cover a range of positive and negative values. The panel consisted of 16 sera from patients with AAV (11 with active disease and 5 in remission), 15 controls with other renal diseases, and 10 healthy controls. The assays were graded positive (red), low positive (Western and IIF), borderline (ELISA) (orange), or negative (green). The figure illustrates the strong concordance among results from the three assays. Sera were considered to have antibodies to LAMP-2 when ≥2 assays were positive. Abbreviations: TX, renal transplant; MGN, membranous nephropathy; IgA GN, IgA nephropathy; MPGN, membranoproliferative GN; TIN, tubulointerstitial nephritis.

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-hLAMP-2, clone H4B4 (Developmental Studies Hybridoma Bank, University of Iowa); polyclonal rabbit ( ; Abnova, Taipei, Taiwan); goat anti-hLAMP-2 (sc-8101; Santa Cruz biotechnology, Santa Cruz, CA); and rabbit anti-rLAMP-2 (Zymed Laboratories, San Francisco, CA).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

    cDNA constructs, generation, and quality control of recombinant hLAMP-2. (A) Representation of cDNA encoding hLAMP-2A with the 28 amino acid leader peptide (LP), 347 amino acid extracellular domain, 24 amino acid transmembrane domain (TM), and 11 amino acid cytoplasmic domain (Cytopl). The two extracellular domain constructs were utilized to express soluble hLAMP-2 in E. coli (hLAMP-2/GST) and mammalian cells (hLAMP-2sol). Both contain the leader peptide but not the transmembrane domain or cytoplasmic tail. hLAMP-2sol expressed in mammalian cells results in an appropriately glycosylated soluble protein exported into the culture supernatant via the default secretory pathway in mammalian cells. The hLAMP-2 cytoplasmic tail contains the signal that directs its retrieval from the plasma membrane to lysosomes. The critical tyrosine was mutated to a histidine in hLAMP-2H (Y/H), which targets it to the plasma membrane when expressed in ldlD cells. (B) Purified hLAMP-2/GST runs as a single 65-kD band on SDS-PAGE and silver stain. Fractions of high purity were pooled and identity of hLAMP-2 was confirmed with an antibody reactive with hLAMP-2 only. (C) Purified hLAMP-2/GST (10 μg/ml) was separated by SDS-PAGE and transferred onto PVDF before probing with specific antibodies to hLAMP-2 (932b), which bound exclusively to the fusion protein. Antibody to GST (anti-GST) recognized both fusion protein and free GST. A human serum (1:100 dilution) containing anti-hLAMP-2 antibodies (Pat. 6) also bound the fusion protein, whereas serum from a healthy control (Healthy Co 50) did not. Serum from a patient with renal disease (Dis. Co 8) contained antibodies to GST. Secondary antibodies alone were negative (anti-human, anti-rabbit). (D) Sensitivity of the Western blot was assessed from doubling dilutions (1:50 to 1:400) of the standard positive control (Stand pos Co) used for all assays. This also confirmed the optimal binding/background ratio was 1:100.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

    doi: 10.1681/ASN.2011090920

    Figure Lengend Snippet: cDNA constructs, generation, and quality control of recombinant hLAMP-2. (A) Representation of cDNA encoding hLAMP-2A with the 28 amino acid leader peptide (LP), 347 amino acid extracellular domain, 24 amino acid transmembrane domain (TM), and 11 amino acid cytoplasmic domain (Cytopl). The two extracellular domain constructs were utilized to express soluble hLAMP-2 in E. coli (hLAMP-2/GST) and mammalian cells (hLAMP-2sol). Both contain the leader peptide but not the transmembrane domain or cytoplasmic tail. hLAMP-2sol expressed in mammalian cells results in an appropriately glycosylated soluble protein exported into the culture supernatant via the default secretory pathway in mammalian cells. The hLAMP-2 cytoplasmic tail contains the signal that directs its retrieval from the plasma membrane to lysosomes. The critical tyrosine was mutated to a histidine in hLAMP-2H (Y/H), which targets it to the plasma membrane when expressed in ldlD cells. (B) Purified hLAMP-2/GST runs as a single 65-kD band on SDS-PAGE and silver stain. Fractions of high purity were pooled and identity of hLAMP-2 was confirmed with an antibody reactive with hLAMP-2 only. (C) Purified hLAMP-2/GST (10 μg/ml) was separated by SDS-PAGE and transferred onto PVDF before probing with specific antibodies to hLAMP-2 (932b), which bound exclusively to the fusion protein. Antibody to GST (anti-GST) recognized both fusion protein and free GST. A human serum (1:100 dilution) containing anti-hLAMP-2 antibodies (Pat. 6) also bound the fusion protein, whereas serum from a healthy control (Healthy Co 50) did not. Serum from a patient with renal disease (Dis. Co 8) contained antibodies to GST. Secondary antibodies alone were negative (anti-human, anti-rabbit). (D) Sensitivity of the Western blot was assessed from doubling dilutions (1:50 to 1:400) of the standard positive control (Stand pos Co) used for all assays. This also confirmed the optimal binding/background ratio was 1:100.

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-hLAMP-2, clone H4B4 (Developmental Studies Hybridoma Bank, University of Iowa); polyclonal rabbit ( ; Abnova, Taipei, Taiwan); goat anti-hLAMP-2 (sc-8101; Santa Cruz biotechnology, Santa Cruz, CA); and rabbit anti-rLAMP-2 (Zymed Laboratories, San Francisco, CA).

    Techniques: Construct, Recombinant, Purification, SDS Page, Silver Staining, Western Blot, Positive Control, Binding Assay

    ANCA and anti-hLAMP-2 antibody titers in patients with AAV. (A and B) Sequential ANCA titers expressed as median and interquartile range in the 43 patients in the Groningen cohort followed for 12 months. ANCA titers decreased rapidly after the induction of treatment. The patients are separated into those who had PR3-ANCA ( n =25; A and C) and MPO-ANCA ( n =18; B and D). (C and D) Sequential titers of antibodies to hLAMP-2 measured by ELISA in the 43 patients in the Groningen cohort followed for 12 months. The upper limit of normal is 29 U and the yellow bar indicates the borderline positive. The patients are separated into those who had PR3-ANCA ( n =25) and MPO-ANCA ( n =18). Anti-hLAMP-2 antibodies became undetectable in

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

    doi: 10.1681/ASN.2011090920

    Figure Lengend Snippet: ANCA and anti-hLAMP-2 antibody titers in patients with AAV. (A and B) Sequential ANCA titers expressed as median and interquartile range in the 43 patients in the Groningen cohort followed for 12 months. ANCA titers decreased rapidly after the induction of treatment. The patients are separated into those who had PR3-ANCA ( n =25; A and C) and MPO-ANCA ( n =18; B and D). (C and D) Sequential titers of antibodies to hLAMP-2 measured by ELISA in the 43 patients in the Groningen cohort followed for 12 months. The upper limit of normal is 29 U and the yellow bar indicates the borderline positive. The patients are separated into those who had PR3-ANCA ( n =25) and MPO-ANCA ( n =18). Anti-hLAMP-2 antibodies became undetectable in

    Article Snippet: The following primary antibodies were used: monoclonal mouse anti-hLAMP-2, clone H4B4 (Developmental Studies Hybridoma Bank, University of Iowa); polyclonal rabbit ( ; Abnova, Taipei, Taiwan); goat anti-hLAMP-2 (sc-8101; Santa Cruz biotechnology, Santa Cruz, CA); and rabbit anti-rLAMP-2 (Zymed Laboratories, San Francisco, CA).

    Techniques: Enzyme-linked Immunosorbent Assay

    PKA but not PKCε plays a role in the expression of Type II priming induced by repeated exposure to CPA A. Male rats that were treated with repeated intradermal administration of CPA (1 µg, hourly, x4) on the dorsum of the hind paw received, 4 days later, an injection of PGE 2 (100 ng) at the same site, in the presence of vehicle (control, black bars) or the PKCε inhibitor PKCε V1-2 (1 µg, white bars), administered 10 min before. Mechanical nociceptive threshold was evaluated 30 min and 4 h after PGE 2 . We observed no difference between the groups in the prolongation of the PGE 2 -induced hyperalgesia ( F 1,10 = 0.27; p = 0.6167, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the prolongation of PGE 2 -induced hyperalgesia in Type II priming produced by CPA is not dependent on PKCε; B ( Left panel ). Rats were treated with intradermal injection of vehicle (control, black bars) or H-89 (1 µg, white bars) on the dorsum of the hind paw. 10 min later, 4 injections of CPA (1 µg, hourly) were performed in both groups of rats. We observed, in the group pretreated with H-89, significant attenuation of the mechanical hyperalgesia induced by the 4 th injection of CPA, when compared with the control group ( F 1,10 = 22.76; *** p

    Journal: Pain

    Article Title: Adenosine-A1 Receptor Agonist Induced Hyperalgesic Priming Type II

    doi: 10.1097/j.pain.0000000000000421

    Figure Lengend Snippet: PKA but not PKCε plays a role in the expression of Type II priming induced by repeated exposure to CPA A. Male rats that were treated with repeated intradermal administration of CPA (1 µg, hourly, x4) on the dorsum of the hind paw received, 4 days later, an injection of PGE 2 (100 ng) at the same site, in the presence of vehicle (control, black bars) or the PKCε inhibitor PKCε V1-2 (1 µg, white bars), administered 10 min before. Mechanical nociceptive threshold was evaluated 30 min and 4 h after PGE 2 . We observed no difference between the groups in the prolongation of the PGE 2 -induced hyperalgesia ( F 1,10 = 0.27; p = 0.6167, two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the prolongation of PGE 2 -induced hyperalgesia in Type II priming produced by CPA is not dependent on PKCε; B ( Left panel ). Rats were treated with intradermal injection of vehicle (control, black bars) or H-89 (1 µg, white bars) on the dorsum of the hind paw. 10 min later, 4 injections of CPA (1 µg, hourly) were performed in both groups of rats. We observed, in the group pretreated with H-89, significant attenuation of the mechanical hyperalgesia induced by the 4 th injection of CPA, when compared with the control group ( F 1,10 = 22.76; *** p

    Article Snippet: The chemicals used in this study were: cordycepin 5’-triphosphate sodium salt (protein translation inhibitor), prostaglandin E2 (PGE2 ; a hyperalgesic agent that directly acts at nociceptors to sensitizes them), N6 -cyclopentyl adenosine (CPA, an A1-adenosine receptor agonist); SU6656 (Src inhibitor), and pertussis toxin (Gi-protein inhibitor), all from Sigma-Aldrich (St. Louis, MO, USA); H-89 dihydrochloride (inhibitor of protein kinase A) and gallein (inhibitor of G- protein β/γ), both from Santa Cruz Biotechnology (Dallas, TX, USA); and PKCεV1–2 (PKCε-I, a PKCε-specific translocation inhibitor peptide) [ ; ], from Calbiochem (La Jolla, CA, USA).

    Techniques: Expressing, Injection, Produced

    Expression of cultured P3 hCECs. Representative sets of photomicrographs showing expression of Na + K + /ATPase and ZO-1 by immunocytochemistry: Immunostaining of Na + K + /ATPase in A : M2 and B : M4. Immunostaining of ZO-1 in C : M2 and D : M4. Control staining E : Isotype matched IgG 1 negative control. ( n = 6; Scale bars = 50 µm).

    Journal: PLoS ONE

    Article Title: Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas

    doi: 10.1371/journal.pone.0028310

    Figure Lengend Snippet: Expression of cultured P3 hCECs. Representative sets of photomicrographs showing expression of Na + K + /ATPase and ZO-1 by immunocytochemistry: Immunostaining of Na + K + /ATPase in A : M2 and B : M4. Immunostaining of ZO-1 in C : M2 and D : M4. Control staining E : Isotype matched IgG 1 negative control. ( n = 6; Scale bars = 50 µm).

    Article Snippet: The following primary antibodies were used: mouse IgG1 anti- Na+ K+ /ATPase α1 (5 µg/mL; Santa Cruz Biotechnology), mouse IgG1 anti-ZO-1 (5 µg/mL; BD Biosciences Pharmingen), and rhodamine conjugated anti-phalloidin (0.5 µM; Invitrogen).

    Techniques: Expressing, Cell Culture, Immunocytochemistry, Immunostaining, Staining, Negative Control

    Activation of caspase-3 by 7-ketocholesterol is reduced in CB2 deficient macrophages. Resident peritoneal macrophages, isolated and pooled from CB2 +/+ and CB2 −/− mice, were cultured for 16 h in the presence or absence of 10 μg/ml 7KC. A: Caspase-3 activity was determined as described in Materials and Methods, and the results are present as the mean fold induction ± SD for three independent experiments. B: Total cell lysates prepared from CB2 +/+ and CB2 −/− MPMs treated with and without 7KC for 16 h were subjected to immunoblotting using antibodies specific for procaspase-3, cleaved caspase-3 and poly-ADP ribose polymerase (PARP). The blots were striped and probed with an Hsc70 antibody to control for equal loading of the gels. The blots shown are representative of three independent experiments. C: The relative amounts of procaspase-3, cleaved caspase-3, full length PARP and cleaved PARP detected by the immunoblots from the three replicate experiments were quantified by densitometric analysis, normalized to Hsc70 levels, and expressed as the mean ratio of cleaved caspase-3 / procaspase-3 ± SD (C) and as the mean ratio of cleaved PARP / total PARP ± SD, respectively (D). * P

    Journal: Journal of Lipid Research

    Article Title: Cannabinoid (CB2) receptor deficiency reduces the susceptibility of macrophages to oxidized LDL/oxysterol-induced apoptosis *

    doi: 10.1194/jlr.M800105-JLR200

    Figure Lengend Snippet: Activation of caspase-3 by 7-ketocholesterol is reduced in CB2 deficient macrophages. Resident peritoneal macrophages, isolated and pooled from CB2 +/+ and CB2 −/− mice, were cultured for 16 h in the presence or absence of 10 μg/ml 7KC. A: Caspase-3 activity was determined as described in Materials and Methods, and the results are present as the mean fold induction ± SD for three independent experiments. B: Total cell lysates prepared from CB2 +/+ and CB2 −/− MPMs treated with and without 7KC for 16 h were subjected to immunoblotting using antibodies specific for procaspase-3, cleaved caspase-3 and poly-ADP ribose polymerase (PARP). The blots were striped and probed with an Hsc70 antibody to control for equal loading of the gels. The blots shown are representative of three independent experiments. C: The relative amounts of procaspase-3, cleaved caspase-3, full length PARP and cleaved PARP detected by the immunoblots from the three replicate experiments were quantified by densitometric analysis, normalized to Hsc70 levels, and expressed as the mean ratio of cleaved caspase-3 / procaspase-3 ± SD (C) and as the mean ratio of cleaved PARP / total PARP ± SD, respectively (D). * P

    Article Snippet: The membranes were blocked in tris buffered saline containing 0.2% Tween-20 (TBS-T) and 5% nonfat dry milk for 1 h followed by overnight incubation at 4°C in TBS-T containing a 1:1000 dilution of a primary antibody to CB2, caspase-3, poly (ADP-ribose) polymerase (PARP) or total Akt1/2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or a 1:5000 dilution of phospho-Akt[Ser473] antibody (Biosource International).

    Techniques: Activation Assay, Isolation, Mouse Assay, Cell Culture, Activity Assay, Western Blot