Journal: Journal of Biological Chemistry

Article Title: ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice*

doi: 10.1074/jbc.m110.108100

Figure Lengend Snippet: FIGURE 2. High dose GW3965 is required for elevated apoE levels in tissue. Cortical and hippocampal extracts from APP/PS1 untreated (: white), APP/PS1 treated (: dark gray), APP/PS1 ABCA1/ untreated (: light gray), and APP/PS1 ABCA1/ treated (: black) mice were immunoblotted for apoE. Left panels are representative blots using undiluted samples from individual mice from the following cohorts: APP/PS1 untreated control: n 7 (cortex), n 8 (hippocampus); APP/PS1 prophylactic: n 8 (cortex) n 8 (hippocam- pus); APP/PS1 low dose therapeutic: n 8 (cortex), n 8 (hippocampus); APP/PS1 high dose therapeutic: n 10 (cortex), n 9 (hippocampus); ABCA1/ APP/PS1 untreated control: n 4 (cortex), n 4 (hippocampus); ABCA1/ APP/PS1 prophylactic: n 8 (cortex) n 8 (hippocampus): ABCA1/ APP/PS1 low dose therapeu- tic: n 7 (cortex), n 6 (hippocampus); ABCA1/ APP/PS1 high dose therapeutic: n 4 (cortex), n 4 (hippocampus). Samples from each mouse were blotted at least twice, normalized to actin, and expressed as fold-difference compared with apoE levels in untreated APP/PS1 controls. Data represent mean S.E., where * represents p 0.05 and ** represents p 0.01 by one-way analysis of variance followed by Tukey post test.

Article Snippet: Western Blot Analysis of ABCA1, ApoE, APP, and APP-CTF— For analysis ofABCA1, apoE, andAPP, 50–100 g of carbonate lysate was electrophoresed through 10% SDS-polyacrylamide gels, transferred to PVDFmembrane (Millipore), and immunodetected using a monoclonal anti-ABCA1 antibody, AC10 (1:1000, a kind gift from Dr. M. R. Hayden), a murine-specific apoE antibody (1:1000, Santa Cruz Biotechnology), an antiAPP antibody that recognizes both murine and human APP (clone 22C11, 1:2000, Chemicon), or an anti-actin antibody (1:5000, Chemicon) as a loading control.

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice*

doi: 10.1074/jbc.m110.108100

Figure Lengend Snippet: FIGURE 3. ABCA1 is required for increased CSF apoE levels in response to GW3965. Equal volumes of CSF from individual mice from untreated control (C), prophylactic (P), low dose therapeutic (T), and high dose therapeutic (TH) groups of APP/PS1 and APP/PS1 ABCA1/ mice were pooled, separated by 6% native PAGE, and immunoblotted for apoE and albumin. Stokes diameter markers are shown on the right.

Article Snippet: Western Blot Analysis of ABCA1, ApoE, APP, and APP-CTF— For analysis ofABCA1, apoE, andAPP, 50–100 g of carbonate lysate was electrophoresed through 10% SDS-polyacrylamide gels, transferred to PVDFmembrane (Millipore), and immunodetected using a monoclonal anti-ABCA1 antibody, AC10 (1:1000, a kind gift from Dr. M. R. Hayden), a murine-specific apoE antibody (1:1000, Santa Cruz Biotechnology), an antiAPP antibody that recognizes both murine and human APP (clone 22C11, 1:2000, Chemicon), or an anti-actin antibody (1:5000, Chemicon) as a loading control.

Techniques: Control, Clear Native PAGE

Journal: ACS Measurement Science Au

Article Title: Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker

doi: 10.1021/acsmeasuresciau.4c00011

Figure Lengend Snippet: DU-DNAzyme walker is activated by UDG and SMUG1. (A) Detection of pure UDG in buffer. (B) Detection of pure SMUG1. (C) Selectivity of the dU-DNAzyme walker for UDG and SMUG1. The concentration of each enzyme was 0.1 U/100 μL. The concentration of BSA tested was 30 μM and IgG was 33 μM. Fluorescence was recorded after 60 min. (D) Uracil DNA glycosylase inhibitor (UGI) inhibits UDG activity and diminishes DNAzyme activation. Fluorescence was recorded after 90 min. Each sample, except for the “No UDG” control, contained 0.1 U of UDG. The “No UDG” control contained 0.1 U of UGI. (E) UDG activity appears to excise uracil, while APE1 and UDG activity dissociates the blocker from the DNAzyme, as visualized with the 10% native PAGE gel. The intermediary band outlined in orange is an unknown structure between the blocked (red) and liberated (yellow) DNAzyme bands. Each reaction mixture contained 50 nM NaCl, 50 mM KCl, 20 mM Tris-acetate (pH 7.9), and the following components: Lane 1, DNAzyme; lane 2, dU blocker; lane 3, blocked DNAzyme; lane 4, blocked DNAzyme + 0.1 U APE1 + 3 mM MgCl 2 ; lane 5, blocked DNAzyme + 0.1 U UDG + 3 mM MgCl 2 ; lane 6, blocked DNAzyme + 0.1 U UDG + 0.1 U APE1 without Mg 2+ ; lane 7, blocked DNAzyme + 0.1 U UDG + 0.1 U APE1 + 3 mM MgCl 2 + 10 mM EDTA; and lane 8, blocked DNAzyme +0.1 U UDG + 0.1 U APE1 + 3 mM MgCl 2 . DZ: DNAzyme. Error bars represent one standard deviation from triplicate experiments.

Article Snippet: APE1, UDG (uracil DNA glycosylase), SMUG1 (single-strand selective monofunctional uracil DNA glycosylase 1), EndoIV (endonuclease IV), AAG (alkyl adenine DNA glycosylase), Fpg (formamidopyrimidine DNA glycosylase), UGI (uracil DNA glycosylase inhibitor), and BSA (bovine serum albumin) were purchased from New England Biolabs (NEB, Whitby, ON, Canada).

Techniques: Concentration Assay, Fluorescence, Activity Assay, Activation Assay, Control, Clear Native PAGE, Standard Deviation

Journal: ACS Measurement Science Au

Article Title: Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker

doi: 10.1021/acsmeasuresciau.4c00011

Figure Lengend Snippet: DU-DNAzyme walker is activated by UDG and SMUG1. (A) Detection of pure UDG in buffer. (B) Detection of pure SMUG1. (C) Selectivity of the dU-DNAzyme walker for UDG and SMUG1. The concentration of each enzyme was 0.1 U/100 μL. The concentration of BSA tested was 30 μM and IgG was 33 μM. Fluorescence was recorded after 60 min. (D) Uracil DNA glycosylase inhibitor (UGI) inhibits UDG activity and diminishes DNAzyme activation. Fluorescence was recorded after 90 min. Each sample, except for the “No UDG” control, contained 0.1 U of UDG. The “No UDG” control contained 0.1 U of UGI. (E) UDG activity appears to excise uracil, while APE1 and UDG activity dissociates the blocker from the DNAzyme, as visualized with the 10% native PAGE gel. The intermediary band outlined in orange is an unknown structure between the blocked (red) and liberated (yellow) DNAzyme bands. Each reaction mixture contained 50 nM NaCl, 50 mM KCl, 20 mM Tris-acetate (pH 7.9), and the following components: Lane 1, DNAzyme; lane 2, dU blocker; lane 3, blocked DNAzyme; lane 4, blocked DNAzyme + 0.1 U APE1 + 3 mM MgCl 2 ; lane 5, blocked DNAzyme + 0.1 U UDG + 3 mM MgCl 2 ; lane 6, blocked DNAzyme + 0.1 U UDG + 0.1 U APE1 without Mg 2+ ; lane 7, blocked DNAzyme + 0.1 U UDG + 0.1 U APE1 + 3 mM MgCl 2 + 10 mM EDTA; and lane 8, blocked DNAzyme +0.1 U UDG + 0.1 U APE1 + 3 mM MgCl 2 . DZ: DNAzyme. Error bars represent one standard deviation from triplicate experiments.

Article Snippet: APE1, UDG (uracil DNA glycosylase), SMUG1 (single-strand selective monofunctional uracil DNA glycosylase 1), EndoIV (endonuclease IV), AAG (alkyl adenine DNA glycosylase), Fpg (formamidopyrimidine DNA glycosylase), UGI (uracil DNA glycosylase inhibitor), and BSA (bovine serum albumin) were purchased from New England Biolabs (NEB, Whitby, ON, Canada).

Techniques: Concentration Assay, Fluorescence, Activity Assay, Activation Assay, Control, Clear Native PAGE, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

doi: 10.1074/jbc.m114.629451

Figure Lengend Snippet: FIGURE 1. Validation of CD44 binding to E-selectin using well established approaches. A, representative flow cytometry histograms of KG1a cells stained for the expression of CD44 (left) and the binding of dE-selectin (middle). A dot plot showing KG1a cells dually stained for CD44 and dE-selectin is shown on the right. Dotted curves show isotype (for CD44) and EDTA (20 mM; for dE-selectin) controls, and shaded curves show specific binding. B, KG1a cells were dually immunostained with CD44mAb(red;left)andrecombinantdE-selectin(green;middle),andinthemergedimage,colocalizationofCD44withdE-selectinisshown(orange;right).Cellsurface labeling with isotype control or secondary antibody alone served as a background control (data not shown). C, CD44 was immunoprecipitated from KG1a cell lysate. The captured proteins were run on a 4–20% SDS-polyacrylamide gradient gel and transferred to a PVDF membrane for Western blot analysis. The membrane was blotted with either dE-selectin (left) or CD44 mAbs (clones 515 and 2C5; right) followed by isotype-matched HRP-conjugated antibody for visualization.D, interaction ofCD44withE-selectinbyblotrollingassaywasperformedonimmunoprecipitatedCD44fromKG1acelllysates.Immunoprecipitatedproteinwasresolvedby4–20% SDS-PAGEandblottedforsLexbindingusingHECA-452mAb.CHO-EcellswerethenperfusedoverthestainedPVDFmembraneat0.25dyne/cm2.Aftercellperfusion, the numbers of rolling cells/field were counted from six experiments using four distinct fields of view in each experiment. As a control, 5 mM EDTA was added to the buffercontainingCHO-Ecells.CHO-Mock(CHO-M)cellswereusedtoassessthenonspecificadhesion.Resultsshownreflectmultipleassays(n4)onHECA-452blots of multiple (n 3) membrane preparations. Data are the mean S.E. (error bars).

Article Snippet: Recombinant mouse E-selectin/human IgG Fc chimera (dE-selectin), recombinant human monomeric E-selectin derived from CHO cells (mE-selectin), recombinant human CD44 (rH-CD44; purified from NSO cells, a mouse myeloma line that lacks the necessary glycosylation machinery to create sLex) (17, 18), and anti-CD44 (2C5, MsIgG2a) were from R&D Systems.

Techniques: Biomarker Discovery, Binding Assay, Flow Cytometry, Staining, Expressing, Labeling, Control, Immunoprecipitation, Membrane, Western Blot, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

doi: 10.1074/jbc.m114.629451

Figure Lengend Snippet: FIGURE 6. SPR binding of CD44 (black line) and PSGL-1 (dashed line) to mE-selectin. A, Hermes-3 mAb (for CD44; 3650 RU), KPL-1 mAb (for PSGL-1; 5000 RU), or isotype control (3400 RU) was used for real time immunoprecipitation from KG1a lysates. Following antigen capture, mE- selectin was injected at either 177 or 354 nM. The sensorgrams were cor- rected for the bulk refractive index and nonspecific interactions using the isotype control. This is a representative experiment of n 2 independent experiments. B, native PAGE of E-selectin monomer and dimer was per- formed where 2 g of mE-selectin and 4 g of dE-selectin were diluted in 2 loading buffer (25% glycerol, 62.5 mM Tris base (pH 6.8), and 1% bro- mphenol blue) and then run on a 10% SDS-polyacrylamide gel without SDS in the running buffer. The gel was then visualized using SimplyBlueTM safe stain. This assay is representative of two independent experiments.

Article Snippet: Recombinant mouse E-selectin/human IgG Fc chimera (dE-selectin), recombinant human monomeric E-selectin derived from CHO cells (mE-selectin), recombinant human CD44 (rH-CD44; purified from NSO cells, a mouse myeloma line that lacks the necessary glycosylation machinery to create sLex) (17, 18), and anti-CD44 (2C5, MsIgG2a) were from R&D Systems.

Techniques: Binding Assay, Control, Immunoprecipitation, Injection, Refractive Index, Clear Native PAGE, Staining

Journal: Cell Reports Methods

Article Title: Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow

doi: 10.1016/j.crmeth.2024.100744

Figure Lengend Snippet:

Article Snippet: Native Human APOE Protein , Abnova , P4281.

Techniques: Clinical Proteomics, Recombinant, Mass Spectrometry, Software