Journal: The Journal of Biological Chemistry

Article Title: Surfactant protein A down-regulates epidermal growth factor receptor by mechanisms different from those of surfactant protein D

doi: 10.1074/jbc.M117.800771

Figure Lengend Snippet: SP-A binds to EGFR in A549 cells, H441 cells, and CHOK1 cells stably expressing EGFR. A, whole-cell lysate of A549 cells was immunoprecipitated (IP) with anti-EGFR monoclonal antibody Ab-11 or control IgG (0. 8 μg) at 4 °C for 16 h. The samples and BSA (200 ng/lane) were subjected to SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with or without SP-A (1 μg/ml) for 16 h. The membranes not incubated with SP-A were subjected to Western blotting using an anti-human EGFR monoclonal antibody (WB: EGFR). The membranes incubated with SP-A were then treated with an anti-human SP-A polyclonal antibody (SP-A pAb) or monoclonal antibodies PE10 (SP-A mAb (PE10)) or PC6 (SP-A mAb (PC6)), which was followed by incubation with HRP-labeled anti-rabbit IgG or anti-mouse IgG (upper panel). As a negative control, the membranes in the absence of incubation with SP-A were also incubated with an anti-human SP-A polyclonal antibody (WB: SP-A pAb) or monoclonal antibodies PE10 (WB: SP-A mAb (PE10)) or PC6 (WB: SP-A mAb (PC6)), which was followed by incubation with HRP-labeled anti-rabbit IgG or anti-mouse IgG (lower panel). The data are representative of three independent experiments. B and C, experimental paradigm described in A was performed in H441 cells (B) and CHOK1 cells stably expressing EGFR (C) by using an anti-human SP-A polyclonal antibody, which was followed by incubation with HRP-labeled anti-rabbit IgG. The data are representative of three independent experiments.

Article Snippet: The H441 human lung adenocarcinoma cell line was obtained from ATCC and maintained in RPMI 1640 medium (Sigma) with 10% (v/v) FCS.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Control, SDS Page, Incubation, Western Blot, Bioprocessing, Labeling, Negative Control

Journal: Journal of medicinal chemistry

Article Title: 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer

doi: 10.1021/acs.jmedchem.8b00838

Figure Lengend Snippet: Inhibition of low molecular weight human uPA by 18 (black) and 27 (red). Data points represent the mean ± SEM (n = 3) from a single representative experiment.

Article Snippet: Experiments with Mouse uPA (active mouse urokinase, HMW, Molecular Innovations Inc., MI, USA) were performed as above with a final enzyme concentration of 20 nM (K m = 580 μ M).

Techniques: Inhibition, Molecular Weight

Journal: Journal of medicinal chemistry

Article Title: 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer

doi: 10.1021/acs.jmedchem.8b00838

Figure Lengend Snippet: Activities of Compounds 4, 5, 18, and 26 against Human and Mouse uPA

Article Snippet: Experiments with Mouse uPA (active mouse urokinase, HMW, Molecular Innovations Inc., MI, USA) were performed as above with a final enzyme concentration of 20 nM (K m = 580 μ M).

Techniques:

Journal: Insect biochemistry and molecular biology

Article Title: Heparan sulfate/heparin glycosaminoglycan binding alters inhibitory profile and enhances anticoagulant function of conserved Amblyomma americanum tick saliva serpin 19

doi: 10.1016/j.ibmb.2016.11.002

Figure Lengend Snippet: List of proteases and their substrates used in substrate hydrolysis assays. Lowest effective protease concentrations (LEPC) and rAAS19 concentrations that inhibit less than 80% of protease activity in specified concentration (IC <80%) are specified.

Article Snippet: The statistical significance of HSGAG binding on rAAS19 inhibitory function was validated using unpaired t-test with Welch's correction in Prism 6 software (GraphPad Software). table ft1 table-wrap mode="anchored" t5 caption a7 Protease (Company) LEPC IC <80% Substrate (Company) Tryptase from Human Lung (Sigma-Aldrich) 4.94 nM NI a N-α-Benzoyl-DL-Arg-pNA (Sigma-Aldrich) Kallikrein from Porcine Pancreas (Sigma-Aldrich) 19.05 nM 1 μM H-D-Pro-Phe-Arg-pNA×2HCl (Chromogenix) Proteinase 3, Human Neutrophil (Athens Research & Technology) 779.31 nM NI a N-Metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Santa Cruz Biotechnology) Elastase from Porcine Pancreas (Sigma-Aldrich) 6.18 nM NI a N-Succinyl-Ala-Ala-Ala-pNA (Sigma-Aldrich) Recombinant Human HMW Urokinase (Molecular Innovations) 9.26 nM NI a CH3SO2-D-CHG-Gly-Arg-pNA×AcOH (Pentapharm) Human tPA, >85% Single Chain (Molecular Innovations) 4.73 nM NI a CH3SO2-D-CHG-Gly-Arg-pNA×AcOH (Pentapharm) Papain (Sigma-Aldrich) 2.14 μM 1 μM N-Benzoyl-Phe-Val-Arg-pNA×HCl (Sigma-Aldrich) Bovine Factor IXa beta (Enzyme Research Laboratories) 31.44 nM 1 μM CH3SO2-D-CHG-Gly-Arg-pNA×AcOH (Pentapharm) Factor Xa Protease (New England Biolabs) 4.65 nM 40 nM Benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNA×HCl (Chromogenix) Human Factor XIa (Enzyme Research Laboratories) 3.69 nM 200 nM H-D-Pro-Phe-Arg-pNA×2HCl (Chromogenix) Human Factor alpha-XIIa (Enzyme Research Laboratories) 2.03 nM 1 μM H-D-Pro-Phe-Arg-pNA×2HCl (Chromogenix) Plasmin from Human Plasma (Sigma-Aldrich) 11.63 nM 20 nM H-D-Val-Leu-Lys-pNA×2HCl (Chromogenix) Thrombin from Bovine Plasma (Sigma-Aldrich) 27.1 nM 100 nM H-D-Phe-Pip-Arg-pNA×2HCl (Chromogenix) Trypsin from Bovine Pancreas (Sigma-Aldrich) 21.01 nM 4 nM Benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNA×HCl (Chromogenix) Open in a separate window a No inhibition List of proteases and their substrates used in substrate hydrolysis assays.

Techniques: Activity Assay, Concentration Assay, Recombinant

Journal: Neuroscience Bulletin

Article Title: Comprehensive Proteomic Profiling of Patients’ Tears Identifies Potential Biomarkers for the Traumatic Vegetative State

doi: 10.1007/s12264-018-0259-x

Figure Lengend Snippet: Gene Ontology analysis revealed 21 proteins involved in response to wounding.

Article Snippet: ELISA and Receiver Operating Characteristic (ROC) Curve Analysis In the verification stage, the levels of 7 promising tear proteins [cystatin B (CTSB), protease, serine 1 (PRSS1), S100 calcium-binding protein A7 (S100A7), glutathione S-transferase P (GSTP1), complement factor H (CFH), kininogen 1 (KNG1), and alpha-1-acid glycoprotein 1 (ORM1)] were measured using the ELISA kits for human CSTB, human PRSS1, human CFH, human KNG1, and human ORM1 (Boster Biological Technology, Wuhan, China) and for human S100A7 and human GSTP1 (from Cloud-clone Corp., Houston, TX).

Techniques:

Journal: Neuroscience Bulletin

Article Title: Comprehensive Proteomic Profiling of Patients’ Tears Identifies Potential Biomarkers for the Traumatic Vegetative State

doi: 10.1007/s12264-018-0259-x

Figure Lengend Snippet: Levels of representative proteins in tears from healthy controls and traumatic vegetative state patients. A List of 7 selected differentially-expressed proteins. Levels of CTSB (B), PRSS1 (C), S100A7 (D), GSTP1 (E), CFH (F), KNG1 (G), and ORM1 (H).

Article Snippet: ELISA and Receiver Operating Characteristic (ROC) Curve Analysis In the verification stage, the levels of 7 promising tear proteins [cystatin B (CTSB), protease, serine 1 (PRSS1), S100 calcium-binding protein A7 (S100A7), glutathione S-transferase P (GSTP1), complement factor H (CFH), kininogen 1 (KNG1), and alpha-1-acid glycoprotein 1 (ORM1)] were measured using the ELISA kits for human CSTB, human PRSS1, human CFH, human KNG1, and human ORM1 (Boster Biological Technology, Wuhan, China) and for human S100A7 and human GSTP1 (from Cloud-clone Corp., Houston, TX).

Techniques: