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: Journal of Controlled Release

Article Title: Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

doi: 10.1016/j.jconrel.2022.05.023

Figure Lengend Snippet: MPLA+PUUC NPs increase T cell responses in the lung when delivered intranasally with spike protein. On days 0 (1st dose) and 28 (2nd dose), female BALB/c mice were immunized I.N. with unformulated (i.e., soluble) or NP-conjugated spike protein (1 μg) and PLGA-PEI NPs (4 mg) loaded with MPLA (24 μg), PUUC (17 μg), and MPLA+PUUC (20 μg, 17 μg). Mice were euthanized and lungs were collected on day 35, one week after the 2nd dose. Lung cells were restimulated with spike peptide pools for 6 h and stained for analysis by flow cytometry. Percentages of cells expressing A) CD4 + CD44 + out of CD45 + cells, B) IFNγ + out of CD4 + CD44 + cells, C) TNFα + out of CD4 + CD44 + cells, D) CD69 + CD103 + (tissue resident memory T cells) out of CD45 + cells. BAL fluid from vaccinated mice using soluble spike antigen was assayed for anti-spike E) IgG and F) IgA with ELISA. G) Sera were assayed for anti-spike IgG with ELISA (error bars represent the SEM). p values are * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 calculated using A – D) One-way ANOVA with Tukey post-hoc test, E , F) Kruskal-Wallis with Dunn's post-hoc test for nonparametric data, or G) Two-way ANOVA with Tukey post-hoc test.

Article Snippet: Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology).

Techniques: Staining, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Controlled Release

Article Title: Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

doi: 10.1016/j.jconrel.2022.05.023

Figure Lengend Snippet: PUUC NPs delivered intramuscularly with spike protein enhance humoral responses. Female BALB/c mice were immunized I.M. into both tibialis anterior muscles at day 0 (1st dose) with soluble spike protein at doses of 80 ng, 200 ng, 1000 ng with or without adjuvant-NPs (4 mg) loaded with PUUC (+P, 20 ng PUUC dose). Peripheral blood was sampled on day 26. On day 28, mice received a 2nd dose of protein subunit vaccines. Mice received the same formulations, except for two groups that received 80 ng spike protein as a 1st dose received 1000 ng spike protein as a 2nd antigen dose (80/1000 and 80/1000 +P). Mice were euthanized on day 36 for to collect blood and popliteal LNs. A) Anti-spike IgG in post-1st dose sera at various dilutions measured by absorbance at 450 nm during ELISA assays and B) comparison of area under the curve (AUC). C – D) Anti-spike IgG in post-2nd dose sera measured by absorbance at 450 nm and comparison of AUC. E) ACE-2 signal measured by absorbance at 450 nm in spike protein neutralization assay with post-2nd dose sera. Absorbance was normalized to a blank well in each row of a 384 well plate to correct for plate effects. Lower absorbance values indicate higher spike-neutralizing antibody levels in sera. Percentages of cells expressing F) Bcl6 + out of B220 + cells, G) GL7 + out of B220 + cells and H) CXCR5 + out of B220 − cells from combined popliteal lymph nodes. B,D) Normality was assessed with the Kolmogorov-Smirnov test. Statistical significance was determined with the Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. E – H) Statistical significance calculated with One-Way ANOVA and Tukey post-hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 for all graphs.

Article Snippet: Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology).

Techniques: Muscles, Adjuvant, Vaccines, Enzyme-linked Immunosorbent Assay, Comparison, Neutralization, Expressing

Journal: Med (New York, N.y.)

Article Title: Omicron neutralizing antibody response following booster vaccination compared with breakthrough infection

doi: 10.1016/j.medj.2022.09.001

Figure Lengend Snippet:

Article Snippet: EIA/RIA high binding ELISA plates , Corning , Ref #359096.

Techniques: Virus, Recombinant, Software, Binding Assay, Enzyme-linked Immunosorbent Assay