Journal: International Journal of Molecular Sciences

Article Title: Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential

doi: 10.3390/ijms21010069

Figure Lengend Snippet: Effects of EDTA on virulence factor production by ( a ) PaM1 and ( b ) PaM5 strains. PaM1 and PaM5 cultures were incubated at 37 °C in the presence or absence of EDTA for 72 h. Following incubation, OD 600 was measured prior the quantification of the virulence factors in culture supernatants (see the Materials and Methods Section for details). Values obtained were normalized by multiplying them by the ratio between OD 600 of the control/OD 600 of the corresponding EDTA-treated samples and reported as mean +/- SEM of three independent experiments. CTRL: bacteria incubated without EDTA; * p

Article Snippet: Determination of minimum inhibitory concentration (MIC) of EDTA towards PaM1 and PaM5 strains was performed according to the standard microdilution method in Muller–Hinton broth (Oxoid) [ ].

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential

doi: 10.3390/ijms21010069

Figure Lengend Snippet: Antibacterial activity of peptide lin-SB056-1, ethylenediaminetetraacetic acid (EDTA), and both in combination against endogenous P. aeruginosa in primary ciliary dyskinesia (PCD) sputum. The effect of lin-SB056-1 and/or EDTA after 1.5 h of incubation in six diluted (1:5) sputum samples was assessed against endogenous P. aeruginosa strains (PaM1 to PaM6) by colony forming unit (CFU) counting. Lin-SB056-1 was tested at 25 μg/mL in combination with 0.625 mM EDTA against PaM1 and PaM5 strains (triangles), and with 1.25 mM EDTA against PaM2, PaM3, PaM4, and PaM6 strains (dots). Control (CTRL): bacteria incubated in diluted sputum only. Individual sputum samples are identified with different colors. Results represent the mean of 6 sputa done in duplicate. Error bars indicate the standard error of the mean. ** p

Article Snippet: Determination of minimum inhibitory concentration (MIC) of EDTA towards PaM1 and PaM5 strains was performed according to the standard microdilution method in Muller–Hinton broth (Oxoid) [ ].

Techniques: Activity Assay, Incubation

Journal: Journal of molecular biology

Article Title: The RelA nuclear localization signal folds upon binding to I?B?

doi: 10.1016/j.jmb.2010.10.055

Figure Lengend Snippet: ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5mM EDTA, and 0.5mM sodium azide

Article Snippet: Two one-liter growths were inoculated with the M9(90% D2 O) culture and induced at OD600 = 0.6 with 0.2 mM IPTG for 24 hours at 18° C. For all IκBα(67-206) preparations, the cells were collected by centrifugation at 5000 rpm for 30 minutes and resuspended in 70 mL/liter of culture of 25 mM Tris (pH7.5), 50 mM NaCl, 0.5 mM EDTA, 10 mM β-mercaptoethanol, 0.3 mM PMSF, and protease inhibitor cocktail (Sigma) and lysed by sonication on ice.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations

doi: 10.1371/journal.pone.0021035

Figure Lengend Snippet: Effect of urea on the intrinsic fluorescence spectra of HsTIM (A and B). Isolation of the two proteins components of HsTIM by size exclusion chromatography (C). In A, the fluorescence spectrum of HsTIM, prepared as described under Material and Methods , was incubated at a concentration of 40 µg/ml in 100 mM triethanolamine, 10 mM EDTA, and 1 mM dithiothreitol (pH 7.4) for 60 min at 25°C with, and without, 6 M urea. At that time the spectra were recorded at an excitation wavelength of 280 nm. B shows the effect of different concentrations of urea on the λmax of its intrinsic fluorescence. C shows the size exclusion chromatography profile (see Methods section) of HsTIM incubated and eluted in a buffer containing 3 M urea. Note that two clearly distinguishable protein peaks were detected; the protein that eluted first was termed P1 and the second P2.

Article Snippet: Generally 300 µl of 20 mM triethanolamine, 0.2 mM EDTA, 200 mM NaCl and 1 mM dithiothreitol (pH 7.4) that contained between 25 to 500 µg of protein were applied and eluted with the same buffer. the columns were calibrated with a gel filtration standard (Bio-Rad) containing the following globular protein markers (molecular mass and retention volumes are reported): thyroglobulin (bovine) (669 kDa, 9.8 ml), γ-globulin (bovine) (158 kDa, 12.9 ml), ovalbumin (chicken) (43 kDa, 15.8 ml), myoglobin (horse) (17 kDa, 17.7 ml), and vitamin B12 (1.35 kDa, 21.1 ml).

Techniques: Fluorescence, Isolation, Size-exclusion Chromatography, Incubation, Concentration Assay

Journal: Journal of Cell Science

Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

doi: 10.1242/jcs.217034

Figure Lengend Snippet: TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P

Article Snippet: Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265).

Techniques: Knock-Out, Incubation, Western Blot, Expressing, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Transfection

Journal: Journal of Cell Science

Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

doi: 10.1242/jcs.217034

Figure Lengend Snippet: Cinnamycin facilitates annexin translocation across membranes in cells. (A) Cinnamycin lipid movement activity. HeLa cells were treated with 1 µM cinnamycin for 50 min at 37°C. Then, recombinant annexin-A5–Cy5 (as a probe for PS) and propidium iodide (PI) (to exclude PI-containing dead cells) were added, and cells were incubated for a further 10 min at 37°C. Annexin-A5–Cy5 binding and PI accumulation were analysed by flow cytometry. Representative histograms of annexin-A5–Cy5 binding to live cells are shown ( n =3). (B) Western blotting analysis of cell lysates and eluates of HeLa cells treated with cinnamycin (30 min at 37°C) and then with EDTA (10 min at 37°C) as indicated. Quantification of cell surface annexin A2 and annexin A5 {fold change measured as band intensity [cinnamycin(eluate/lysate)/DMSO(eluate/lysate)]} is shown. Results are mean±s.e.m. from n =3 biological replicates; * P

Article Snippet: Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265).

Techniques: Translocation Assay, Activity Assay, Recombinant, Incubation, Binding Assay, Flow Cytometry, Cytometry, Western Blot

Journal: eLife

Article Title: Type XVII collagen coordinates proliferation in the interfollicular epidermis

doi: 10.7554/eLife.26635

Figure Lengend Snippet: COL17 distribution is modulated by aPKC. ( a ) COL17 staining of whole IFE skin treated with 5 mM EDTA. The quantitative fluorescent intensity of COL17 in basal cells of IFE from control (PBS) and 5 mM EDTA treated (n = 4). Mann-Whitney test. Samples were taken from young adult WT mice at 6–10 weeks. Scale bar: 20 μm. ( b ) Phospho-aPKC labeling (indicated with arrows) and quantitative fluorescent intensity results from young and aged WT IFE skin (n = 4). Mann-Whitney test. Scale bar: 20 μm.( c ) Representative figures of asymmetric cell division (ACD; scored as perpendicular to basement membrane) and symmetric cell division (SCD; in parallel to basement membrane) in young IFE. Survivin staining indicates the direction of the cell division. Laminin β1 signifies basement membrane. Scale bar: 10 μm. Graph of percentage of ACD and SCD in young and aged IFE (n = 4). Student’s t-test. ( d–e ) The pharmacological inhibition of pan-aPKC (d, 1 μM of Go6983; 0.00002% DMSO as control) and aPKCλ/ζ (e, 10 μM of myr PSI; water as control) in whole IFE skin from young adult WT mice, followed by COL17 staining. The quantitative fluorescent intensity of COL17 in lateral membrane of basal cells from control and 1 μM Go6983 treated ( d ) and 1 μM myr PSI ( e ) (n = 4). Mann-Whitney test. Scale bar: 10 μm. BM, basement membrane. ( f–h ) EDTA treatment (5 mM; PBS as control, f ) and pharmacological inhibition of pan-aPKC (g, 1 μM of Go6983; 0.00002% DMSO as control) and aPKCλ/ζ (h, 10 μM of myr PSI; water as control) on 3D epidermis. The relative fluorescent intensity of COL17 in lateral membrane of basal cells was measured (n = 4). Mann-Whitney test. BM, basement membrane. Scale bar: 20 μm. The data are the means ± SE. *0.01

Article Snippet: Ex vivo inhibitor treatment Whole mice paw skin samples were treated with the following inhibitors for 1 hr at 4°C: EDTA, pan-PKC inhibitor Go6983 (Tocris Bio, Bristol, UK) and myristoylated pseudosubstrate Inhibitor (myr PSI; Calbiochem, Billerica, Massachusetts, USA) before staining.

Techniques: Staining, MANN-WHITNEY, Mouse Assay, Labeling, Inhibition

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Human Osteoclasts are Inducible Immunosuppressive Cells in Response to T cell-Derived IFN-γ and CD40 Ligand in vitro

doi: 10.1002/jbmr.2294

Figure Lengend Snippet: The suppression of OCs on T-cell proliferation is IFN-γ and CD40L dependent Proliferation of CD4 + T cells stimulated by (A) α-CD3/CD28 Dynabeads at a T/Bead ratio of 2:1 or (B) allogeneic DCs at a T/DC ratio of 10:1 in Transwells in the absence or presence of autologous OCs with IFN-γ neutralizing or IFNGR1 blocking antibodies (20 μg/mL) for 4–6 d. (C) Proliferation of CD4 + T cells stimulated by allogeneic DCs under the contact coculture of autologous OCs with IFN-γ neutralizing, CD40 or CD40L blocking antibodies (20 μg/mL). (D) Expression of IFNGR1 and CD40 on OCs, as measured by flow cytometry. OCs coculutred with α-CD3/CD28 Dynabeads-activated CD4 + T cells in Transwell system were harvested by Trypsin/EDTA detachment, stained by antibodies and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.

Article Snippet: To detect the viability of OCs during the coculture with α-CD3/CD28 DynaBeads-stimulated T cells in Transwells, coculutred OCs were digested from the culture plate with Trypsin/EDTA, and stained by using LIVE/DEAD® Fixable Dead Cell Stain Kit (Molecular Probes, Life Technologies).

Techniques: Blocking Assay, Expressing, Flow Cytometry, Cytometry, Staining

Journal: Scientific Reports

Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

doi: 10.1038/srep29077

Figure Lengend Snippet: Effect of SAP on heat-induced GST aggregation in MES-Ca and Tris-EDTA buffers. ( a,c ) Time course of GST aggregation monitored by turbidity in the absence (black diamond) or presence of 1:10 (molar ratio of SAP to GST) (orange circle), 1:5 (red circle), or 1:2 (dark brown circle) SAP in MES-Ca buffer ( a ), or 1:10 (right green circle), 1:5 (green circle), or 1:2 (dark green circle) SAP in Tris-EDTA buffer ( c ) at 43 °C. Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b,d ) Turbidity of each sample at 32 h in MES-Ca ( b ) and Tris-EDTA buffers ( d ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. **P

Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

Techniques:

Journal: Scientific Reports

Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

doi: 10.1038/srep29077

Figure Lengend Snippet: Effects of CRP and SAP on Aβ(1-40) amyloid fibril formation in Ca 2+ -free Tris-EDTA buffer. ( a ) Time course of fibril formation monitored by ThT fluorescence in the absence (black diamond) or presence of 1:500 (molar ratio of CRP to Aβ(1-40)) (light blue square), 1:100 (blue square), or 1:20 (dark blue square) CRP, or 1:500 (orange circle), 1:100 (red circle), or 1:20 (dark brown circle) SAP. Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b ) ThT fluorescence of each sample at 156 h in ( a ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. *P

Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

doi: 10.1038/srep29077

Figure Lengend Snippet: Structure and assembly states of CRP and SAP. ( a,b ) Structures of pentameric CRP and SAP. These figures were prepared with the PDB files 1B09 for CRP ( a ) and 1SAC for SAP ( b ) using the software PyMOL. Pentamers were viewed along the 5-fold axis of symmetry from the A face (upper) and perpendicular to the 5-fold axis (lower). Yellow: calcium ions. ( c–f ) Analysis of molecular weight distribution of CRP and SAP by gel filtration chromatography. CRP ( c,d ) and SAP ( e,f ) at 1.5 μM were incubated in Tris-EDTA (pH 7.5) ( c,e ), Tris-Ca (pH 7.5) ( d ), or MES-Ca buffer (pH 7.0) ( f ) at 37 °C for 0 (black line) or 72 h (red line), then 300 μL aliquots were applied on a column equilibrated and eluted with the same buffer at 15 °C. Elution was monitored by absorbance at 280 nm. Arrows in each figure indicate the elution volumes of molecular weight markers (kDa).

Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

Techniques: Software, Molecular Weight, Filtration, Chromatography, Incubation

Journal: Scientific Reports

Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

doi: 10.1038/srep29077

Figure Lengend Snippet: Interactions of CRP and SAP with fresh and aggregated Aβ(1-40) and D76N β2-m. ( a–f ) Binding of Aβ(1-40) ( a–c ) and D76N β2-m ( d–f ) to immobilized CRP and SAP. CRP and SAP were immobilized on an ELISA plate and incubated with fresh ( f ) and aggregated (ag) Aβ(1-40) and D76N β2-m in Tris-EDTA ( a,d ), Tris-Ca ( b,e ), or MES-Ca buffer ( c,f ). The data are mean ± SD of three independent incubations. Representative data of three independent experiments are shown. ( g,h ) Crosslinking experiments. At 0 h (fresh mixture, f) and at the beginning of the growth phase (aggregated mixture, ag), the reaction mixture containing Aβ(1-40) or D76N β2-m was spiked with 1:20 CRP and SAP, and incubated for 30 min at 37 °C. After BS 3 was added to the mixture, SDS-PAGE (top) and western blotting analysis (bottom) were performed. In western blotting analysis, bound Aβ(1-40) and D76N β2-m were detected with anti-human Aβ(1-40) and β2-m antibodies, respectively.

Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, SDS Page, Western Blot

Journal: Current Chemical Genomics

Article Title: Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

doi: 10.2174/1875397301105010030

Figure Lengend Snippet: Tolerance of GAPDHS assay to DMSO. DMSO was tested in the GAPDHS HTS assay (30 nM GAPDHS, 50 mM glycine, 50 mM potassium phosphate, pH 8.9, 5 mM EDTA, 1 mM DTT, 1% DMSO, 0.5 mM NAD and 0.5 mM GAP) at the indicated concentrations of DMSO.

Article Snippet: Cells were harvested, resuspended in lysis buffer (DPBS, 1 mM PMSF (MP Biomedicals, Solon, OH), 1 mM EDTA (Promega, Madison, WI), 1 mM DTT (US Biochemicals, Santa Clara, CA), 10 µg/ml lysozyme (Fisher BioReagents, Suwanee, GA), 0.1% Triton X-100) and lysed at 23,000 psi by passage through a M-110EH homogenizer (Microfluidics, Newton, MA).

Techniques: HTS Assay

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: 2. Relationship to Aβ immunotherapy

doi: 10.1016/j.jalz.2017.04.015

Figure Lengend Snippet: Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to NHS to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. EDTA treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.

Article Snippet: NHS plus 10 mM EDTA (Amresco, Solon, OH) (final concentration in the serum) was employed as a control.

Techniques: Activation Assay, Western Blot, Generated, Molecular Weight

Journal: International Journal of Biological Sciences

Article Title: An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

doi:

Figure Lengend Snippet: Preparation of HDL associated LCAT. Size exclusion chromatography was perfo rmed at ambient temperature using a Superose-6 10/300 column on a Bio-Rad FPLC system (Hercules, CA). A 200 µl aliquot of plasma was injected per run, and eluted in PBS with 1 mM EDTA at a flow rate of 0.2 ml/min. Total cholesterol in each fraction was measured using the Cholesterol E Kit. LCAT activity was determined as following: A. 20 µl of each fraction of human plasma were incubated at 37ºC for 5 h; B. 2.5 µl of each fraction of WT mouse plasma were incubated at 37ºC for 2 h; C. 10 µl of each fraction of apoE-null mouse plasma were incubated at 37ºC for 4 h.

Article Snippet: Preparation of HDL associated LCAT enzyme Fresh human or mouse blood was drawn into tubes containing potassium EDTA (Becton Dickinson, Franklin lakes, NJ), and plasma was separated immediately by low-speed centrifugation at 2500 rpm (1430g ) for 30 min at 4ºC.

Techniques: Size-exclusion Chromatography, Fast Protein Liquid Chromatography, Injection, Flow Cytometry, Activity Assay, Incubation

Journal: eLife

Article Title: Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

doi: 10.7554/eLife.34798

Figure Lengend Snippet: Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida -BFP hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. ( A ) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. ( B ) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. ( C ) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: ( D ) E-cadherin-GFP or ( E ) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both ( D ) and ( E ), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. ( F ) RAW-Dectin1 cells were allowed to internalize C. albicans -hyphae in the presence or absence of 4 mM EDTA. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.

Article Snippet: Where indicated, cells were treated with either vehicle or 1 μM Latrunculin A (Sigma-Aldrich) after 1 hr Candida- BFP infection, or pretreated 30 min with 4 mM EDTA (Bioshop), followed by infection with Candida- BFP in the presence of EDTA.

Techniques: Incubation, Staining, Immunostaining, Expressing, Transfection, Confocal Microscopy

Journal: Nature Communications

Article Title: Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure

doi: 10.1038/ncomms8229

Figure Lengend Snippet: N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes. In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. ( a ) Chemical structure of SERCA2a SUMOylation activator. N106, N -(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. ( b-g ) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant ( τ ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n =25 cardiomyocytes from each three of hearts per condition. ( h ) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM KCl, 5 mM MgCl 2 , 1 mM EDTA at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. ( i ) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. ( j ) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. ( n =3 mice hearts per each condition). Data are represented as mean±s.e.m. * P ≤0.05; ** P

Article Snippet: The ATPase activity were measured at 25 °C in 50 mM MOPS (Boston Bioproducts, MA, USA), 100 mM KCl (Boston Bioproducts), 5 mM MgCl2 (Boston Bioproducts), 1 mM EDTA (Boston Bioproducts) at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM.

Techniques: Activity Assay, In Vitro, Isolation, Negative Control, Fluorescence, Immunoprecipitation, Western Blot, Mouse Assay