Journal: Cancer Informatics

Article Title: A Pan-Cancer Analysis of Alternative Splicing Events Reveals Novel Tumor-Associated Splice Variants of Matriptase

doi: 10.4137/CIN.S19435

Figure Lengend Snippet: Flow cytometric analysis reveals surface expression of matriptase splice variants. Cells were transfected with 10 μg of empty vector alone (pTT5) or 5 μg of each matriptase variant plus 5 μg of HAI-1 ( A – G ). The next day, duplicate wells containing 100,000 cells per well were stained with either human anti-matriptase or mouse anti- SPINT1 (HAI-1) antibodies (data not shown) followed by species-specific secondary Alexa Fluor ® 647 Goat anti-IgG-Fc antibodies plus the live/dead cell discriminator 7-AAD followed by flow cytometric analysis. The gating tree is as follows: ( A ) SSC versus FSC depicts the distribution of cells as oppose to the debris that was excluded; to ( B ) living cells not stained with 7-AAD. ( C ) Wild-type matriptase, ( D ) matriptase variant A1, and ( E ) matriptase variant A3 ( F ) graph depicting the mean fluorescent intensity plus/minus the standard error of mean of matriptase expressed on the surface of CHO cells. These data are representative of three independent experiments analyzed with a student’s t -test ( P < 0.05). Flow cytometry data were acquired on an IntelliCyt ® HTFC, which uses an Accuri ® C6 Flow Cytometer ® (BD Biosciences) with the sip time set at three seconds. Laser lines for this instrument are 488 and 640 nm. FL3 emission detection for 7-AAD is >670 nm, and FL4 emission detection for Alexa Fluor ® 647 is 675/25 nm. ( G ) Recombinant wild type, A1, and A3 variants were immunoprecipitated with 1.5 μg of human anti-matriptase antibody, followed by Western blot analysis on the clarified start lysates (20 μg each) and elutions (15 μL each). The arrow shows the bands corresponding to the expected size of each matriptase variant.

Article Snippet: Data were acquired with an Intelli-Cyt ® high-throughput flow cytometer (HTFC) that consisted of an Accuri ® C6 Flow Cytometer ® (BD Biosciences), CFlow ® Software (version 1.0.227.4), HyperCyt ® CFlow Automator (version 3.4.0.0), and HyperView iDM ® Client Edition 4.0 (R2 version 4.0.4395).

Techniques: Expressing, Transfection, Plasmid Preparation, Variant Assay, Staining, Flow Cytometry, Recombinant, Immunoprecipitation, Western Blot

Journal: PLoS Neglected Tropical Diseases

Article Title: A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

doi: 10.1371/journal.pntd.0002423

Figure Lengend Snippet: Neutralization of pseudovirus bearing CHIKV S27 wild-type (black); CHIKV A226V mutant (light blue); SFV (red); SINV (magenta); RRV (green) and VSV (dark blue) envelope by ( A ) C9 or ( B ) E8 mAbs. Antibody concentration is shown in the x-axis. The results are expressed as the percentage of no antibody control and represent mean of triplicate wells, and is representative of three experiments.

Article Snippet: Cells were stained for 1 h with human mAbs C9 (0.42 μg/ml), E8 (2 μg/ml), CKV061 (0.75 μg/ml, isolated from phage display library in identical manner to E8), E26D9.02 (0.5 μg/ml, a gift from Dendritics), and rabbit polyclonal antibody (1∶2000 dilution, a gift from IBT Bioservices) diluted in 10% NGS (Sigma). mAbs were detected using 3.75 μg/ml AlexaFluor488-conjugated secondary antibody in 10% NGS (Jackson ImmunoResearch Laboratories) for 1 h. Mean cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (HTFC, Intellicyt).

Techniques: Neutralization, Mutagenesis, Concentration Assay, Control

Journal: PLoS Neglected Tropical Diseases

Article Title: A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

doi: 10.1371/journal.pntd.0002423

Figure Lengend Snippet: ( A ) Summary of antibody/antigen interactions. Binding of anti-CHIKV antibody C9 to intact CHIKV VLPs was detected using the FoteBio OctetRed biosensor. C9 mAb (66.6 nM) binding to CHIKV VLPs or a non-particle surface control is used to show binding specificity of mAb to intact CHIKV VLP. ( B ) C9 dose response curve for binding intact CHIKV VLPs. Raw data curves for antibody associating and dissociating from captured CHIKV VLPs are shown in black and fitted curves are shown in red. Data were fitted to a 1∶1 binding model to determine association rate (k on ) and dissociation rate (k off ), and equilibrium binding affinity (K D ) was calculated. C9 binds CHIKV VLPs with 1.21 nM apparent affinity.

Article Snippet: Cells were stained for 1 h with human mAbs C9 (0.42 μg/ml), E8 (2 μg/ml), CKV061 (0.75 μg/ml, isolated from phage display library in identical manner to E8), E26D9.02 (0.5 μg/ml, a gift from Dendritics), and rabbit polyclonal antibody (1∶2000 dilution, a gift from IBT Bioservices) diluted in 10% NGS (Sigma). mAbs were detected using 3.75 μg/ml AlexaFluor488-conjugated secondary antibody in 10% NGS (Jackson ImmunoResearch Laboratories) for 1 h. Mean cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (HTFC, Intellicyt).

Techniques: Binding Assay, Control

Journal: PLoS Neglected Tropical Diseases

Article Title: A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

doi: 10.1371/journal.pntd.0002423

Figure Lengend Snippet: C57BL/6 mice (n = 4 mice per group) were injected with (i) PBS; (ii) purified C9 mAb; or (iii) purified control human mAb at 0.5 mg/mouse by the intraperitoneal route one day (day −1) prior to infection on day 0 with CHIKV (isolate LR2006-OPY1). ( A ) Peripheral blood viremia (CCID 50 /ml). X-axis represents days post-CHIKV inoculation; ( B ) Foot swelling over time is presented as a group average of the percentage increase in foot height×width (in the metatarsal region) for each foot compared with the same foot on day 0 (n = 8 feet).

Article Snippet: Cells were stained for 1 h with human mAbs C9 (0.42 μg/ml), E8 (2 μg/ml), CKV061 (0.75 μg/ml, isolated from phage display library in identical manner to E8), E26D9.02 (0.5 μg/ml, a gift from Dendritics), and rabbit polyclonal antibody (1∶2000 dilution, a gift from IBT Bioservices) diluted in 10% NGS (Sigma). mAbs were detected using 3.75 μg/ml AlexaFluor488-conjugated secondary antibody in 10% NGS (Jackson ImmunoResearch Laboratories) for 1 h. Mean cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (HTFC, Intellicyt).

Techniques: Injection, Purification, Control, Infection

Journal: PLoS Neglected Tropical Diseases

Article Title: A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

doi: 10.1371/journal.pntd.0002423

Figure Lengend Snippet: C57BL/6J neonatal mice were injected with 5×10 5 PFU CHIKV (isolate S27) virus. Co-incident with infection, groups of mice (n = 5 to 8 mice per group) were inoculated with C9 mAb at the indicated concentrations or human IgG as control. The protective nature of C9 mAb at different concentrations is represented as Kaplan-Meier survival curves.

Article Snippet: Cells were stained for 1 h with human mAbs C9 (0.42 μg/ml), E8 (2 μg/ml), CKV061 (0.75 μg/ml, isolated from phage display library in identical manner to E8), E26D9.02 (0.5 μg/ml, a gift from Dendritics), and rabbit polyclonal antibody (1∶2000 dilution, a gift from IBT Bioservices) diluted in 10% NGS (Sigma). mAbs were detected using 3.75 μg/ml AlexaFluor488-conjugated secondary antibody in 10% NGS (Jackson ImmunoResearch Laboratories) for 1 h. Mean cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (HTFC, Intellicyt).

Techniques: Injection, Virus, Infection, Control

Journal: PLoS Neglected Tropical Diseases

Article Title: A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

doi: 10.1371/journal.pntd.0002423

Figure Lengend Snippet: Therapeutic protection against CHIKV-driven lethality in neonate mice 1 .

Article Snippet: Cells were stained for 1 h with human mAbs C9 (0.42 μg/ml), E8 (2 μg/ml), CKV061 (0.75 μg/ml, isolated from phage display library in identical manner to E8), E26D9.02 (0.5 μg/ml, a gift from Dendritics), and rabbit polyclonal antibody (1∶2000 dilution, a gift from IBT Bioservices) diluted in 10% NGS (Sigma). mAbs were detected using 3.75 μg/ml AlexaFluor488-conjugated secondary antibody in 10% NGS (Jackson ImmunoResearch Laboratories) for 1 h. Mean cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (HTFC, Intellicyt).

Techniques: Control