Journal: Cell Death & Disease

Article Title: ErbB4 signaling stimulates pro-inflammatory macrophage apoptosis and limits colonic inflammation

doi: 10.1038/cddis.2017.42

Figure Lengend Snippet: The metalloprotease TACE/ADAM17 and γ -secretase are necessary for NRG4-induced macrophage apoptosis. ( a ) Schematic model of potential ErbB4 signaling in macrophages following ligand binding. (Step 1) Extracellular receptor cleavage by ADAM17; (Step 2) intracellular cleavage by γ -secretase and generation of ErbB4 intracellular domain (4ICD); (Step 3) migration of the active signaling fragment 4ICD to various intracellular compartments. ( b ) Classically activated BMDMs were pre-treated for 1 h with metalloprotease inhibitor (GM6001, 10 μ M), γ -secretase inhibitor (DAPT, 10 μ M), or ADAM17 inhibitor (GW280264X, 3 μ M) followed by 100 ng/ml NRG4 and 100 ng/ml LPS. Percent cell viability was analyzed by rezasurin-based cell titer assay ( n =5 independent experiments). ( c ) Immunofluorescence analysis of ErbB4 localization to the mitochondria of classically activated BMDMs treated with or without NRG4 (100 ng/ml) for 48 h. Arrows point to representative cells with ErbB4/mitochondrial overlap (images representative from n =4 independent experiments). ( d ) Western blot analysis for the ErbB4 4ICD fragment and COX IV (mitochondrial marker) of isolated cytoplasmic and mitochondrial fractions of classically activated BMDMs treated with NRG4 for 48 h. Representative blots from n =3 independent experiments shown. ( e ) Classically activated BMDMs were treated with or without NRG4 for 48 h, stained with fluorescent cationic dye (MitoCapture), and analyzed by flow cytometry for red (aggregated dye in healthy mitochondria) and green (cytoplasmic dye resulting from mitochondria with disrupted membrane potential) fluorescence. Fold change in green/red ratio is shown ( n =5 independent experiments). ( f ) NRG4-induced killing of classically activated macrophages was assessed in the presence of inhibitors to necroptosis (necrostatin-1) or apoptosis (NS3694). Each panel, n =5 independent experiments. Error bars represent S.E.M. * P <0.005; ** P <0.01; *** P <0.001

Article Snippet: In some experiments cells were then pre-treated for 30 min with 2 μ g/ml ErbB4 neutralizing antibody (Millipore, Billerica, MA, USA, 05–478) before incubation with 100 ng/ml NRG4 (Reprokine, Valley Cottage, NY, USA) for 1 h then LPS (100 ng/ml) for 48 h. Some cells were pre-treated with the metalloprotease inhibitor GM6001 (Tocris, Minneapolis, MN, USA, #2983) at 10 μ M, γ -secretase inhibitor DAPT (Tocris, #2634) at 10 μ M, ADAM17 inhibitor GW280264X at 3 μ M (Aobious, Inc., Gloucester, MA, USA), RIPK1 inhibitor, Necrostatin-1 at 50 μ M (Cayman Chemical, Ann Arbor, MI, USA, 11658), or caspase-9 activation inhibitor, NS3694 at 10 μ M (Millipore, 178494) prior to NRG4 and LPS treatment as described in Results.

Techniques: Ligand Binding Assay, Migration, Titer Assay, Immunofluorescence, Western Blot, Marker, Isolation, Staining, Flow Cytometry, Membrane, Fluorescence

Journal: Biosensors

Article Title: Study of Immobilization Procedure on Silver Nanolayers and Detection of Estrone with Diverged Beam Surface Plasmon Resonance (SPR) Imaging

doi: 10.3390/bios3010157

Figure Lengend Snippet: Experimental reflectivity vs . internal angle, θ , using the set-up depicted in : circles—after incubation of the sample in 10 mM of 11-MUA for 24 h; diamonds—after immobilization of rabbit anti-estrone polyclonal IgG 1:10; triangles—after adding 0.0042 gr estrone in 1 mL DI. Calculated approximations for each signal are shown as smooth curves.

Article Snippet: 11-Mercapto-undecanoic acid (11-MUA) 95%, 45056-1 Sigma Aldrich; dimethyl sulfoxide (DMSO) (Merck, Germany); ethanol 97%; N,N ' -dicyclohexylcarbodiimide (DCC); EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) GR1-SI-E, 1769-0005 (Sigma-Aldrich Co., Israel); luminescence HRP antibody and luminol (Sigma-Aldrich Co., Israel); N-hydroxysuccinimide (NHS) GR1-FL-56480-0100 (Sigma-Aldrich Co., Israel); Phosphate buffered saline (PBS) pH 7.4; estrone E9750 (Sigma-Aldrich Co., Israel); rabbit anti-estrone polyclonal IgG 7030-0604 (AbD Serotec Co.); silicon adhesive isolators 3560-25EA (Sigma-Aldrich Co., Israel).

Techniques: Incubation

Journal: Biosensors

Article Title: Study of Immobilization Procedure on Silver Nanolayers and Detection of Estrone with Diverged Beam Surface Plasmon Resonance (SPR) Imaging

doi: 10.3390/bios3010157

Figure Lengend Snippet: Reflected images from DBSPRI sensor, top: 68 nm Ag on BK7 glass after incubation in ( a ) 10 mM 11-MUA for 24 h and ( b ) after incubation in 10 mM 11-MUA for 24 h immobilized rabbit anti-estrone polyclonal IgG, while adding NHS + DCC prior to the immobilization; bottom: analyzed images in Radon space.

Article Snippet: 11-Mercapto-undecanoic acid (11-MUA) 95%, 45056-1 Sigma Aldrich; dimethyl sulfoxide (DMSO) (Merck, Germany); ethanol 97%; N,N ' -dicyclohexylcarbodiimide (DCC); EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) GR1-SI-E, 1769-0005 (Sigma-Aldrich Co., Israel); luminescence HRP antibody and luminol (Sigma-Aldrich Co., Israel); N-hydroxysuccinimide (NHS) GR1-FL-56480-0100 (Sigma-Aldrich Co., Israel); Phosphate buffered saline (PBS) pH 7.4; estrone E9750 (Sigma-Aldrich Co., Israel); rabbit anti-estrone polyclonal IgG 7030-0604 (AbD Serotec Co.); silicon adhesive isolators 3560-25EA (Sigma-Aldrich Co., Israel).

Techniques: Incubation