Journal: PLoS ONE

Article Title: A Subregion of Reelin Suppresses Lipoprotein-Induced Cholesterol Accumulation in Macrophages

doi: 10.1371/journal.pone.0136895

Figure Lengend Snippet: (A) ELISA plates were coated with c-myc peptide, purified R5-6 or R5-6C, or null eluent from empty-vector transformed bacteria as a control (Ctrl), and then incubated with RAW 264.7 cell lysates. The apoER2 and VLDLR in RAW 264.7 cell lysates bound to ELISA plates were detected using antibodies against apoER2 and VLDLR. (B) RAW 264.7 cells were transfected with empty pcDNA3.1B vector or apoER2-GFP expression plasmid, and then incubated with 30 μg/ml of apoB-carrying and apoE-free lipoproteins (+LP) or without lipoproteins (-LP) in the presence or absence of 0.2 μg/ml purified R5-6 protein. The amount of R5-6 bound to the cell surface was determined by ELISA using an antibody against c-myc epitope. The background binding was determined using a normal mouse IgG. (C-D) RAW 264.7 cells were treated with 0.2 μg/ml of purified R5-6 protein or culture medium alone (Ctrl). The protein level of ABCA1 was determined by immunoblotting and quantified relative to β-actin. (E) RAW 246.7 cells were treated as described in plane B. The mRNA level of ABCA1 was determined by quantitative real-time RT-PCR and normalized relative to GAPDH mRNA. (F) RAW 264.7 cells were labeled with 0.25 μCi/ml of 3 H-cholesterol, followed by incubation with 5 μg/ml c-myc peptide, 0.2 μg/ml purified R5-6 protein or vehicle control (Ctrl). Cholesterol efflux was determined in cells incubated with or without apoAI treatment, and was expressed as the percentage of radioactivity in the medium compared to the total radioactivity in the cells and medium. ApoAI-mediated cholesterol efflux was calculated as the difference of efflux from cells in the presence and absence of apoAI treatment. Data represent the mean ± SEM of four or more independent experiments. * p < 0.05 vs . Ctrl; † , p <0.05 vs . c-myc-immobilized ELISA plates or treated cells; ‡ , p < 0.05 vs . cells transfected with the same plasmids and untreated with R5-6; $ , p < 0.05 vs . cells transfected with empty vector and treated with R5-6; and # , p < 0.05 vs . cells transfected with apoER2-GFP-expression plasmid and untreated with lipoproteins.

Article Snippet: PI3K inhibitor LY294002 (sc-201426), Sp1 inhibitor mithramycin A (sc-200909), ELISA plate (sc-204463), scrambled siRNA, and siRNAs specific for VLDLR or apoER2, antibodies against ABCA1 (sc-58219), β-actin (sc-47778), apoER2 (sc-20746), VLDLR (sc-18824), and mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Plasmid Preparation, Transformation Assay, Bacteria, Control, Incubation, Transfection, Expressing, Binding Assay, Western Blot, Quantitative RT-PCR, Labeling, Radioactivity

Journal: Frontiers in Allergy

Article Title: Immunotherapy with biodegradable nanoparticles encapsulating the oligosaccharide galactose-alpha-1,3-galactose enhance immune tolerance against alpha-gal sensitization in a murine model of alpha-gal syndrome

doi: 10.3389/falgy.2024.1437523

Figure Lengend Snippet: Prophylactic treatment with NP-αGal prevents IgE-mediated allergic responses to αGal. ( A ) Schematic of prophylactic NP treatment in a mouse model of tick extract-induced IgE sensitization to αGal. AGKO mice received two doses of NP-αGal–HSA or control NP-HSA, 2 weeks apart, prior to intradermal injections (i.d.) of whole-body protein extract prepared from lone star seed ticks supplemented with 50 μg αGal-BSA on day 0 and identically prepared booster injections on days 7 and 31. On day 35, mice received an intragastric challenge with beef extract and were analyzed 1.5 h later. ( B ) Concentration of MCPT-1 in the sera of mice after the beef extract challenge measured by ELISA. ( C ) Fold increase in histamine released from the basophil activation test (BAT) with cetuximab measured by ELISA. ( D ) Serum levels of total IgE, tick-specific IgE, and αGal-specific IgE, IgG1, and IgG2b from mice on day 35 measured by ELISA or Luminex. All data are expressed as mean ± SEM. The results shown are representative of two independent experiments. P = *0.05, **0.01, and ****0.0001, or ns = not significant, with an unpaired, two-tailed t -test.

Article Snippet: For αGal-specific IgG1 and IgG2b ELISAs, high-binding plates were coated with 3 μg/mL αGal polymer (GlycoTech) in PBS, and serum was diluted at 1:50 and 1:200.

Techniques: Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Luminex, Two Tailed Test

Journal: Frontiers in Allergy

Article Title: Immunotherapy with biodegradable nanoparticles encapsulating the oligosaccharide galactose-alpha-1,3-galactose enhance immune tolerance against alpha-gal sensitization in a murine model of alpha-gal syndrome

doi: 10.3389/falgy.2024.1437523

Figure Lengend Snippet: Therapeutic administration of NP-αGal reduces hypersensitivity responses in mice following an intragastric challenge with beef extract. ( A ), Schematic of therapeutic NP treatment. AGKO mice received intradermal injections (i.d.) of whole-body protein extract prepared from lone star seed ticks supplemented with 50μg αGal-BSA on day 0 and identically prepared booster injections on days 7 and 31. On days 33 and 47, mice were intravenously given NP-αGal–HSA or control NP-HSA and, 2 weeks later, were given a booster of tick extract. On day 68, mice received an intragastric challenge with beef extract and were analyzed 1.5 h later. ( B ) Concentration of MCPT-1 in the sera of mice after a beef extract challenge measured by ELISA. ( C ) Fold increase in histamine released from the basophil activation test (BAT) with cetuximab measured by ELISA. ( D ) Serum levels of total IgE, tick-specific IgE, and αGal-specific IgE, IgG1, and IgG2b from mice on day 68 measured by ELISA or Luminex. All data are expressed as mean ± SEM. P = *0.05, and ****0.0001, or ns = not significant, with an unpaired, two-tailed t -test.

Article Snippet: For αGal-specific IgG1 and IgG2b ELISAs, high-binding plates were coated with 3 μg/mL αGal polymer (GlycoTech) in PBS, and serum was diluted at 1:50 and 1:200.

Techniques: Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Luminex, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A High-throughput Assay for Phosphoprotein-specific Phosphatase Activity in Cellular Extracts *

doi: 10.1074/mcp.O112.024059

Figure Lengend Snippet: A high-throughput phosphatase assay that monitors endogenous activity toward specific phosphoprotein substrates. A, recombinant phosphorylated substrate is adsorbed onto a polystyrene microtiter plate, the immobilized phosphosubstrate are treated with PPase lysate, and the dephosphorylation reaction is halted with the addition of a phosphatase inhibitor mixture. B, the remaining phosphosubstrate is labeled with anti-phosphoprotein primary antibody, biotinylated secondary antibody, and streptavidin-HRP for colorimetric detection. Phosphatase activity is determined by the drop in phosphoprotein signal in lysate-treated samples relative to negative controls.

Article Snippet: High-throughput PPase Assay High protein-binding polystyrene microtiter plates (Corning Costar, Lowell, MA) were coated overnight with the indicated amounts of recombinant phosphorylated MAPK.

Techniques: High Throughput Screening Assay, Phosphatase Assay, Activity Assay, Recombinant, De-Phosphorylation Assay, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A High-throughput Assay for Phosphoprotein-specific Phosphatase Activity in Cellular Extracts *

doi: 10.1074/mcp.O112.024059

Figure Lengend Snippet: ELISA-based quantification of recombinant pMAPKs adsorbed to 96-well plates. A–C, Twofold dilution series (relative level 1.0 = ∼400 ng) of pERK (A), pJNK (B), and pp38 (C) were adsorbed onto polystyrene microtiter plates and detected by ELISA using phospho-specific antibodies. The detection saturates at ∼75 ng for pERK (A), ∼400 ng for pJNK (B), and ∼75 ng for pp38 (C). Arrows indicate the quantity of pMAPKs used in the PPase assay. Data are shown as the mean ± S.E. of three independent assay replicates.

Article Snippet: High-throughput PPase Assay High protein-binding polystyrene microtiter plates (Corning Costar, Lowell, MA) were coated overnight with the indicated amounts of recombinant phosphorylated MAPK.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Virology

Article Title: Diversity of the murine antibody response targeting influenza A(H1N1pdm09) hemagglutinin

doi: 10.1016/j.virol.2014.04.011

Figure Lengend Snippet: Binding of rmAb to inactivated vaccines and hemagglutination inhibition assays rmAb produced as described were screened by ELISA for reactivity with TIV (containing CA/07(H1pdm), VIC/210(H3), and BR/60(B)) and with the individual monovalent inactivated vaccine (MIV) constituents: CA/07(H1pdm), VIC/210(H3), and BR/60(B). Data are shown as minimum rmAb concentration (ng/ml) required to produce a 2-fold signal above negative control. “-“, no reactivity seen at concentrations ≤25 μg/mL. “NT”, not tested. rmAb were also tested in HI assays against CA/07(H1pdm), VIC/210(H3), and B/BR/60. Values are shown as minimum concentration (ng/mL) rmAb required to inhibit 4 HA units of antigen. “*“, no HI activity seen at concentrations ≤12.5 μg/mL.

Article Snippet: ELISA Costar Hi Bind plates (Corning Inc., Tewksbury, MA) were coated overnight with TIV or the individual monovalent inactivated virus constituents at 4 °C.

Techniques: Binding Assay, Vaccines, HI Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control, Activity Assay