Journal: The Journal of Biological Chemistry

Article Title: Internal Tandem Duplication Mutations in FLT3 Gene Augment Chemotaxis to Cxcl12 Protein by Blocking the Down-regulation of the Rho-associated Kinase via the Cxcl12/Cxcr4 Signaling Axis *

doi: 10.1074/jbc.M114.568287

Figure Lengend Snippet: ITD-FLT3-induced enhancement of cell migration toward Cxcl12 does not involve up-regulation of Cxcr4 signaling. A, baseline migration of ITD-FLT3− or ITD-FLT3+ (N51) Ba/F3 cells was quantified for 24 h (left panel, *, p < 0.05 compared with ITD-FLT3− Ba/F3 cells, n = 3). The right panel indicates the dose-dependent inhibition of the migration of ITD-FLT3+ (N51) Ba/F3 cells using CEP701 or AC220. The fold-change in migration relative to that of the DMSO control is shown (*, p < 0.05, n = 3). B, migration of ITD-FLT3+ (N51 and N78) and ITD-FLT3− Ba/F3 cells toward 100 ng/ml Cxcl12 (upper panel in the left panel). Cell migration was normalized to the number of input cells that were incubated during the corresponding time frame. (*, p < 0.05 compared with the ITD-FLT3− Ba/F3 cells, n = 2.) The lower panel on the left shows the migration of ITD-FLT3+ (N51) and ITD-FLT3− Ba/F3 cells toward various concentrations of Cxcl12 for 4 h. (*, p < 0.05 compared with the ITD-FLT3− Ba/F3 cells, n = 2.) The center panel represents the percentage of migration of ITD-FLT3+ Ba/F3 cells toward 100 ng/ml Cxcl12 in the presence of 1 or 5 nm AC220. (*, p < 0.05 compared with control cells in the absence of AC220, n = 3.) The right panel shows the fold-change in migration of ITD-FLT3+ MV4-11 cells toward 100 ng/ml CXCL12 in the presence of 1.0 nm CEP701 or 5.0 nm AC220 compared with the DMSO control. (*, p < 0.05, n = 3.) C, left panel shows the migration of ITD-FLT3+ (N51) and ITD-FLT3− Ba/F3 cells toward 10 ng/ml Cxcl12 in the presence of 1 nm AC220 and/or 10 μg/ml of a neutralizing anti-mouse CXCR4 antibody. The right panel shows the migration of the same cells toward 100 ng/ml CXCL12 in the presence of 20 μm AMD3100 and/or 10 nm CEP701. (*, p < 0.05 compared with the ITD-Flt3− cells treated with DMSO and CXCL12; †, p < 0.05 compared with the ITD-Flt3+ cells treated with DMSO and CXCL12, n = 3.) D, left panel, expression of Cxcr4 in ITD-FLT3+ (N51 and N78) Ba/F3 cells compared with ITD-FLT3− Ba/F3 cells analyzed via flow cytometry. The relative mRNA level of Cxcr4 was determined using an mRNA array. The right histogram shows Cxcr4 expression ITD-FLT3+ (N51 and N78) Ba/F3 cells in the presence or absence of a 4-h stimulation with 100 ng/ml Cxcl12. Quantification of the Cxcr4 surface protein and mRNA expression levels is shown below the plots. The data shown represent one of the three experiments. Right panel, left bar graph shows a comparison of the CXCR4 mRNA levels between patients with ITD-FLT3+ AML (78 cases) and ITD-FLT3− AML (190 cases). The CXCR4 levels were analyzed using three CXCR4 probes, which were deposited in a public gene expression database (GSE1159, www.ncbi.nlm.nih.gov) (26) (*, p < 0.05). The right bar graph indicates the relative surface expression of CXCR4 in MV4-11 cells that express endogenous ITD-FLT3 compared with RS4;11 cells, which do not harbor ITD-FLT3, as determined via flow cytometry (*, p < 0.05, n = 3). E, calcium mobilization in the ITD-FLT3+ (N51) and ITD-FLT3− Ba/F3 cells stimulated with 10 or 100 ng/ml Cxcl12. The fold-increase in fluorescence following stimulation with Cxcl12 compared with before incubation is shown (*, p < 0.05, n = 3). F, IL-3-deprived ITD-FLT3− and ITD-FLT3+ Ba/F3 cells (1, N51; 2, N73; and 3, N78) were incubated in 100 ng/ml Cxcl12 for 10 min. Erkp42/p44 and Creb phosphorylation was determined via Western blot analyses before and after incubation in Cxcl12. The signal of the protein was measured and analyzed using the densitometer in the Odyssey infrared imaging system (LI-COR biosciences, Lincoln, NE). The relative expression level of each phosphorylated protein compared with total protein is shown below the blots.

Article Snippet: The antibodies against phosphorylated myosin phosphatase-targeting subunit 1 (p-Mypt1), total Mapk p42/p44 , phosphorylated MAPK p42/p44 , total Creb, and phosphorylated Creb were purchased from Cell Signaling Technology (Danvers, MA). mRNA Microarray Analyses and RT-PCR Ba/F3 cells expressing wild-type FLT3 or ITD- FLT3 (N51, N73, and N78) obtained from three different patients with AML were provided by Dr. D. G, Gilliland of Harvard Medical School ( 22 ).

Techniques: Migration, Inhibition, Incubation, Expressing, Flow Cytometry, Fluorescence, Western Blot, Imaging

Journal: Molecular Cancer Research

Article Title: R-spondin2 Suppresses the Progression of Hepatocellular Carcinoma via MAPK Signaling Pathway

doi: 10.1158/1541-7786.mcr-19-0599

Figure Lengend Snippet: Figure 5. MAPK phosphorylation was upregulated in R-spondin2–overexpressing cells and downregulated in R-spondin2–knockdown cells. A, Heat map of differentially expressed genes between R-spondin2–overexpressing and control Huh-7 cells using Agilent Whole Human Genome Oligo Microarray. The data were accessible through GEO Series accession number GSE152354 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE152354). B, Cluster analysis showing that the MAPK signaling was significantly increased after transfection. C and D, Effects of R-sopndin2 overexpression and knockdown on MAPK phosphorylation, according to Western blot assay.

Article Snippet: The nitrocellulose membrane was then incubated using antibodies for R-spondin2, N-cadherin (Santa), b-catenin, ERK, phosphor(p)-ERK, Akt, p-Akt, c-Jun, p-c-Jun, p38-MAPK, p-p38MAPK (all Cell Signaling Technology), and b-actin (Sigma).

Techniques: Phospho-proteomics, Knockdown, Control, Microarray, Transfection, Over Expression, Western Blot

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: ( A ) Magnitude and kinetics of plasma IgG to A244 D11 gp120 (vaccine strain boost immunogen) measured by BAMA in 70 RV305 participants at RV144 weeks 0 (pre-vaccination) and 26 (2 weeks post final RV144 vaccination) and RV305 weeks 0 (RV305 baseline; time point of first RV305 boost), 2 (two weeks post RV305 first boost), 24 (time point of second RV305 boost), 26 (two weeks post second RV305 boost), 48 (6 months post second boost), and 72 (1 year post second boost). IgG BAMA response magnitude is expressed as mean fluorescence intensity (MFI) after blank bead subtraction (MFI-Blank). Red, Combination group (ALVAC-HIV + AIDSVAX B/E) (n = 20 vaccinees); green, AIDSVAX B/E only group (n = 18 vaccinees); blue, ALVAC-HIV only group (n = 19 vaccinees); black, RV305 placebo group (RV144 vaccinees administered RV305 placebo) (n = 13 participants). Boxplots depict the median (midline) and 25 th and 75 th percentiles, with the colored symbols indicating the response for a single participant measured at a 1:50 dilution. Open gray triangles indicate negative responders. Gray lines connect the response from a single participant between time points. Response rates at each time point are shown at the top of each plot.( B ) Log 2 fold difference in post second boost (RV305 week 26) / post first boost (RV305 week 2) plasma IgG binding to linear epitopes in C1.2, V2 hotspot (V2.hs), V3, and C5.2 within AE.A244 and AE.TH023 gp120 assessed by peptide microarray mapping assay. Horizontal bar pointing to the left of the x = 0 line (solid black vertical line) indicates a higher response magnitude measured at RV305 week 2 compared to RV305 week 26; horizontal bar pointing the right indicates a higher response magnitude measured at RV305 week 26 versus RV305 week 2. ( C ) Log 2 fold difference in Combination (ALVAC-HIV + AIDSVAX B/E) group / AIDSVAX B/E only group plasma IgG binding to linear epitopes in C1.2, V2.hs, V3, and C5.2 within AE.A244 gp120 and AE.92TH023 gp120 assessed by peptide microarray mapping assay. Horizontal bar pointing to the left of the x = 0 line at indicates a higher response magnitude measured in the AIDSVAX B/E only group; horizontal bar pointing to the right indicates a higher response magnitude measured in the Combination group. ( D ) Response rate and magnitude of post first boost (RV305 week 2) and post second (RV305 week 26) boost plasma IgG binding to linear AE.A244 V2.hs. The number of positive responders is shown over the total number of individuals analyzed at each time point, represented as a bar graph displaying percent responders. Box plots depict the median (midline) and 25 th and 75 th percentiles, with the colored symbols indicating the epitope mapping response magnitude for a single participant. ( E ) Response rate and magnitude of plasma IgG binding to AE.A244 V3 linear peptide. ( F ) Prevalence of CD4-induced (CD4i) IgG antibodies among RV305 participants. Differential binding plots displaying BAMA MFI-Blank values for IgG binding to YU2 gp120 WT (y-axis) and YU2 gp120 I420R mutant (x-axis) proteins at RV144 week 26 and RV305 weeks 0, 2, and 26. The diagonal dashed gray line indicates a wild-type to mutant binding ratio of 2.5 (cut-off for positivity). The CD4-induced (CD4i) monoclonal antibody 17b was used as a positive control for YU2 gp120 WT/I420R differential binding. Colored symbols represent positive responders with differential binding ratios of ≥ 2.5, indicating the presence of CD4i specificities. Response rate (percent responders over the total number of participants analyzed) is shown at the top of each plot.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Fluorescence, Binding Assay, Peptide Microarray, Mapping Assay, Mutagenesis, Positive Control

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: ( A ) Longitudinal plasma IgG binding antibody responses to AE.A244 V1V2 tags (V1V2 from RV144 vaccine boost–A244 gp120) and a panel of 16 geographically and genetically diverse gp70 V1V2 scaffold proteins representing global HIV-1 diversity determined by BAMA. Group median MFI among positive responders is plotted for each strain, color-coded by HIV-1 subtype; blue, clade A; red, CRF01_AE; purple, clade B; gray, CRF01_BC, green, clade C. Dotted black horizontal line, showing MFI equal to 100 indicates the minimum threshold for positivity for individual samples. ( B ) BAMA IgG breadth scores for binding to the gp70 V1V2 breadth panel. Scores were calculated based on averaging the mean fluorescence intensities (MFIs) of the individual antigens in the panel. Each symbol represents the breadth score for a single vaccine recipient. Red, Combination group (ALVAC-HIV + AIDSVAX B/E); green, AIDSVAX B/E only group; blue, ALVAC-HIV only group. Boxplots depict the median (midline) and 25 th and 75 th percentiles for the Combination (ALVAC-HIV/AIDSVAX B/E) and AIDSVAX B/E only groups. Differences in breadth scores between RV144 and RV305 post boost time points was assessed using the two-sided Wilcoxon Signed Rank test, combining data for Combination and AIDSVAX B/E only groups . ( C ) Magnitude-breadth (MB) plot showing the number of antigens in the V1V2 panel (n = 16) with positive binding (breadth) (y-axis) at a given response magnitude (log10 binding antibody MFI) (x-axis) among positive responders in the Combination (ALVAC-HIV + AIDSVAX B/E) and AIDSVAX B/E only groups. Dashed lines display MB curves for each individual plasma sample measured at RV144 week 26 (turquoise), RV305 week 2 (pink), and RV305 week 26 (orange). Solid bold lines show the median MB among positive responders at each immunization time point. AUC values summarize the MB at a given time point across the entire range of MFI values. ( D ) Plasma IgG concentrations to V1V2 antigens associated with RV144 vaccine efficacy extrapolated by 4-parameter logistic (4-PL) regression of V2-specific monoclonal antibody CH58 standard curve titrations run in each BAMA. Concentrations are plotted in μg/mL for positive responders in each group across the studied immunogenicity time points, with each dot representing the concentration for a single plasma sample. The midline of the box plot denotes the median concentration, and the ends of the box plot denote the 25 th and 75 th percentiles among positive responses. (E) Durability of V1V2 IgG responses. Fold decline in binding V1V2 IgG MFI between 2 weeks after the last RV305 boost (week 26) to 12 months post last boost (week 72). Results are presented as log 2 fold change, with the midline of the box plots indicating median and ends of the box plots indicating the 25 th and 75 th percentiles. The whiskers denote the minimum and maximum data points no more than 1.5 times the interquartile range (IQR). Black dots represent data points that lie outside of the median ± 1.5 times the IQR. Criteria for the fold (wk26/wk72) calculation: 1) response is positive at week 26, 2) MFI < 23000 at week 26, 3) MFI > 100 at week 72. Antigens with greater than or equal to 6 data points meeting this criteria for both the Combination (ALVAC-HIV/AIDSVAX B/E) and AIDSVAX B/E only groups are plotted for each vaccine boost regimen. Proximity of the bar to the y-axis indicates better durability.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Binding Assay, Fluorescence, Immunopeptidomics, Concentration Assay

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: ( A ) Kinetics of plasma IgG responses in RV305 vaccine recipients to vaccine strain (92TH023 gp120, A244 gp120, and MN gp120) and gp120 (n = 8) and gp140 (n = 8) Env breadth panel antigens representing genetic and geographic HIV-1 diversity. Group median BAMA MFI binding values among positive responders, color coded by HIV-1 subtype, are shown for two RV144 and 6 RV305 sampling time points; blue, clade A; red, CRF01_AE; purple, clade B; gray, CRF01_BC, green, clade C. ( B ) BAMA IgG breadth scores to the gp140 Env breadth panel at RV144 and RV305 post boost time points. Box and whisker plots show the median and interquartile ranges of scores across the Combination and AIDSVAX B/E only groups. Comparison of median breadth scores (aggregated for the Combination and AIDSVAX B/E only groups) across post RV144 boost (week 26) and RV305 boost time points (weeks 2 and 26) were performed using the two-sided Wilcoxon Signed Rank Test . ( C ) Magnitude-breadth plot of IgG binding antibody responses to the gp140 breadth panel among Combination and AIDSVAX B/E only positive responders at 2 weeks post final RV144 vaccination (week 26) and 2 weeks post first and second RV305 boosts (weeks 2 and 26). Breadth is defined as the proportion of antigens in the 8 antigen gp140 breadth panel (y-axis) with log10 (MFI-Blank) greater than the threshold on the x-axis. Dashed lines display MB curves for each individual plasma sample measured at RV144 week 26 (turquoise), RV305 week 2 (pink), and RV305 week 26 (orange). Solid bold lines show the median MB among positive responders at each immunization time point. AUC values summarize the MB at a given time point across the entire range of MFI values. ( D ) Durability of Env gp140 IgG responses. Fold decline in IgG antibody binding magnitude to gp140 antigens from two weeks post last RV305 boost (week 26) to 12 months post last boost (week 72). Results are presented as log2 fold change, with the midline of the box plots indicating median and ends of the box plots indicating the 25th and 75th percentiles. The whiskers denote the minimum and maximum data points no more than 1.5 times the interquartile range (IQR). Data points that lie outside of the median ± 1.5 times the IQR are shown as black dots. Criteria for the fold (wk26/wk72) calculation: 1) response is positive at week 26, 2) MFI < 23000 at week 26, 3) MFI > 100 at week 72. Antigens with greater than or equal to 6 data points meeting this criteria for both the Combination (ALVAC-HIV/AIDSVAX B/E) and AIDSVAX B/E only groups are plotted for each vaccine boost regimen. Proximity of the bar to the y-axis indicates better durability. ( E ) Magnitude-breadth scores across C1.2, V2 hotspot (V2.hs), V3, and C5.2 linear epitopes in gp120 calculated as weighted means, using a hierchical clustering tree method (R package “mdw”) for binding to all strains with a positivity rate of >20% for any time point for each epitope. Box plots depict the median (midline) and 25th and 75th percentiles, with each symbol, color coded by group, indicating the epitope mapping breadth score for a single participant.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Binding Assay, Sampling, Whisker Assay, Comparison

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: Plasma from a subset of 70 RV305 recipients was tested for IgG1-IgG4 subclass binding to ( A ) vaccine strain immunogens (92TH023, A244 gp120, MN gp120), gp120 (n = 8) and gp140 (n = 8) Env breadth panel and ( B ) V1V2 (n = 16) breadth panel antigens and to AE.A244 V1V2 tags. Group median BAMA MFI is shown at each time point. Dotted black horizontal line, showing MFI equal to 100 indicates the minimum threshold for positivity for individual samples.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Binding Assay

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: (A) Response rate (top panel) and response magnitude (bottom panel) for HIV-1 envelope-specific IgG3 in plasma at two weeks post final RV144 vaccination (week 26) and two weeks post first and second RV305 boosts (weeks 2 and 26, respectively) against four vaccine-matched antigens (A244 D11 gp120, MN gp120, 92TH023 gp120, and AE.A244 V1V2 tags) identified as primary immune variables. The number of positive responders is shown over the total number of individuals analyzed at each time point, represented as a bar graph displaying percent responders. Box plots depict the median (midline) and 25 th and 75 th percentiles, with the colored symbols indicating the BAMA IgG3 response magnitude for a single participant expressed as MFI. Open gray triangles depict negative responders. ( B ) IgG3 breadth scores, defined as the mean of IgG3 responses to Env gp120, gp140, and gp70 V1V2 breadth panel antigens (panel size = 8, 8, and 16 antigens, respectively), are shown for RV144 week 26 and RV305 weeks 2 and 26. Each symbol represents the breadth score for a single participant. Red, Combination (ALVAC-HIV + AIDSVAX B/E) group; green, AIDSVAX B/E only group; blue, ALVAC-HIV only group. Box plots show the distribution of breadth scores among Combination and AIDSVAX B/E only boost recipients, displaying the median (midline), 25 th and 75 th percentiles. ( C ) Magnitude breadth (MB) plots of IgG3 binding antibody responses to the gp70 V1V2 breadth panel (top plot), gp120 breadth panel (middle plot), and gp140 breadth panel (bottom plot) measured at RV144 week 26 (turquoise), RV305 week 2 (pink), and RV305 week 26 (orange) for positive responders that received ALVAC-HIV + AIDSVAX B/E or AIDSVAX B/E only boosts. The y-axes depicts breadth (number of antigens in a given panel with positive response, depicted as a percentage) at a given response magnitude (log 10 binding antibody MFI) across the x-axes. Sample-specific and group averaged MB curves are represented by dashed and solid lines, respectively. AUC values summarize the MB profile at a given time point across the entire range of binding values across the x-axis. ( D ) Concentration of IgG3 antibodies to three V1V2 antigens associated with decreased HIV-1 risk in RV144 plotted for BAMA positive responders by vaccine group across longitudinal RV144 and RV305 time points. V2-specific microgram per milliter (μg/ml) equivalent concentrations for each plasma sample were quantified by 4 parameter logistic (4PL) regression based on standard curve titration of the IgG3 V2-specific monoclonal antibody CH58 curves run in each BAMA. Each symbol represents the concentration for a single plasma sample. The midline of the box plot denotes the median concentration, and the ends of the box plot denote the 25 th and 75 th percentiles among positive responses.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Binding Assay, Concentration Assay, Titration

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: ( A ) HIV-1-specific plasma IgG4 levels to vaccine-matched gp120 Envelope (A244 D11 gp120, MN gp120, 92TH023 gp120) and V1V2 (AE.A244 V1V2 tags) primary antigens determined by BAMA. Response rates (top panel) and binding magnitudes (bottom panel) are plotted for each group at two weeks post final RV144 vaccination (week 26) and two weeks post first and second RV305 boosts (weeks 2 and 26, respectively). Box plots (bottom panel) denote the median (midline) and interquartile ranges among positive responses. Solid dots depict positive responders, and open gray triangles depict non responders. ( B ) BAMA analysis of plasma IgA1 subclass levels in 32 randomly selected RV305 vaccinees (n = 12, 8, and 12 in groups 1, 2, and 3, respectively) against a panel of 5 Envelope proteins encompassing vaccine immunogens (A244 D11 gp120, MN gp120, 92TH023 gp120), 00MSA gp140, and clade A consensus (A.con.env03 gp140) proteins. Response rates (top panel) and binding magnitudes (bottom panel) are plotted for each group at two weeks post final RV144 vaccination (week 26) and two weeks post first and second RV305 boosts (weeks 2 and 26, respectively). Box plots (bottom panel) denote the median (midline) and interquartile ranges among positive responses. Solid dots depict positive responders, and open gray triangles depict non responders.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Binding Assay

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: Plasma antibody-mediated uptake of ( A ) Con S gp140 or ( B ) A244 gp120-conjugated fluorescent beads in THP-1 cells was quantified by flow cytometry for vaccinees in the down-selected set of 70 RV305 participants. Top panels in A and B show the number of responders over the total number of participants analyzed whereas bottom panels denote the mean phagocytosis score per group at RV144 peak immunogenicity (week 26), RV305 baseline (week 0) and post boost time points (weeks 2 and 26). The box plots show the distribution of positive responses, with the mid-line denoting the median and the ends of the boxplot denoting the 25 th and 75 th percentiles. Data are representative of two independent experiments. ( C ) Phagocytosis AUC was calculated for a subset of 38 vaccinees (n = 14, 11, and 13 in groups 1, 2, and 3, respectively) whose plasma was tested for Con S gp140 ADCP. AUC for ADCP scores from a 5 point, 5-fold dose response curve from 50 μg/ml to 0.08 μg/ml is shown. ( D ) Spearman correlations between Con S gp140 phagocytosis AUC and IgG1 or IgG4 Con S gp140 BAMA MFI (aggregated for Combination and AIDSVAX B/E only groups) at RV305 week 2 and week 26; two-sided P values are shown for Spearman’s rho correlation coefficients. IgG2 and IgG3 binding levels were too low to perform correlation analysis. ( E ) Representative histograms of HIV-1 CM235 Tomato virion internalization by primary monocytes in the presence of Ab8367, a gp120-specific native IgG3 monoclonal antibody (mAb) isolated from a RV305 vaccinee by antigen-specific single memory B cell sorting. Ab8367 IgG1 and IgG3 recombinant mAbs mediated phagocytosis, showing greater potency when expressed as IgG3 (middle panel) compared to IgG1 (right panel). Red lines represent antibody-mediated internalization of virions; black lines represent internalization of virions in the absence of antibody. The shaded gray region represents the negative control in the absence of virus. The influenza hemagglutinin (HA)-specific broadly neutralizing antibody CH65 IgG3 was used as a negative control. Data are representative of two independent experiments. ( F ) Purified plasma IgG from 15 RV305 vaccine recipients (n = 5 in each group) was tested for antibody-mediated HIV-1 92TH023 -Tomato virion internalization in primary monocytes, analyzed across RV144 and RV305 baseline and immunogenicity time points by flow cytometry. Each solid line, color coded by vaccination group, represents one vaccinee. The horizontal dashed black line denotes the threshold for positivity. Response rates at each time point are shown at the top of the graph. ( G ) HIV-1 92TH023 infectious virion capture was measured in 15 RV305 vaccinees (n = 5 in each group) using purified IgG from longitudinal plasma samples. The horizontal dashed black line represents the threshold for positivity. Each solid line represents one vaccinee. The percentage of vaccinees with vaccine-elicited antibodies capable of infectious virion capture is shown at the top of the graph for each time point.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Clinical Proteomics, Flow Cytometry, Immunopeptidomics, Binding Assay, Isolation, FACS, Recombinant, Negative Control, Virus, Purification

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: The fold change in magnitude of humoral and cellular immune measurements (n = 54) from 2 weeks post first boost (week 2) to 2 weeks post second boost (week 26) was calculated for each participant (n = 70) and then log 10 transformed. Measurements were grouped by generalized features (BAMA IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, linear peptide microarray binding, CD4 and CD8 T cell polyfunctionality, neutralizing antibody (nAb), ADCC, or ADCP effector functions) indicated by the gray bars on the right. The boxplots span the 25 th percentile to the 75 th percentile, with a black bar at the median. Whiskers extend from the greater of the minimum data point and median—1.5 times the interquartile range (IQR) to the lesser of the maximum data point and median + 1.5 times the IQR, where IQR = 75 th -25 th percentile. Data points that lie outside of the median ± 1.5 times the IQR are shown as black dots. Horizontal bar pointing to the left of the x = 0 line indicates a higher response magnitude measured at RV305 week 2 compared to RV305 week 26; horizontal bar pointing to the right of the x = 0 line indicates a higher response magnitude measured at RV305 week 26 compared to RV305 week 2.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Transformation Assay, Peptide Microarray, Binding Assay

Journal: PLOS Pathogens

Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response

doi: 10.1371/journal.ppat.1011359

Figure Lengend Snippet: Heatmap of 28 immune response variables longitudinally analyzed at RV144 peak immunogenicity (week 26) and two weeks post first and second RV305 boosts (weeks 2 and 26, respectively). All data was log10-transformed and then each variable [measurement type (BAMA MFI, BAMA breadth score, and ADCP score)] was scaled across participant, visit, antigen, and in the case of BAMA isotype/subclass for vaccinees in the down-selected set of 70 RV305 participants. The median of the scaled values are plotted by visit and treatment group, ordered by assay type [BAMA (IgG, IgG1, IgG3, IgG4, IgA, IgA1), and antigen-conjugated bead antibody-dependent phagocytosis (ADCP)]; scaling precludes comparison of variables with each other, but variables can be compared across groups and visits. Columns designate immunogenicity time points whereas rows represent immune measurements. Color intensity is directly proportional to response magnitude, with the darker colors indicating higher magnitudes and the lighter colors indicating lower magnitudes.

Article Snippet: HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen).

Techniques: Immunopeptidomics, Transformation Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Identification of the minimal human P2P. ( A ) Schematic representation of P2P 5′-truncations and their cloning strategy as used in this study. The translational start site is designated ‘+1’. ( B ) Regulatory DNA regions of the human PHD2 gene were cloned into luciferase reporter vectors that were transiently transfected into human U2OS osteosarcoma cells. One day after transfection, cells were incubated for 24 h at 20 or 0.2% O 2 . Hypoxic IF (mean values ± SD) of relative luciferase activities were calculated from three independent experiments performed in triplicates. Mutation of a single HBS (black rectangles in A) completely abrogated hypoxic inducibility of all constructs. ( C ) HeLa and U2OS cells were incubated at 20 or 0.2% O 2 for 4–24 h and protein levels of HIF-1α, PHD2 and β-actin were analyzed by immunoblotting. Total RNA was isolated from cultures treated as in (B) and mRNA levels of PHD2 and CA9 were determined by RT-qPCR. Transcript levels of CA9 served as positive control to confirm continuous hypoxic responses. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (±SD).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Cloning, Clone Assay, Luciferase, Transfection, Incubation, Mutagenesis, Construct, Western Blot, Isolation, Quantitative RT-PCR, Positive Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Hypoxic transactivation of the P2P by ETV4 requires HIF-1α activity. ( A ) Standard dual luciferase reporter gene assays of seven reevaluated hits from the transcription factor overexpression array. Wild-type (left panel) or HBS mutant (right panel) P2P regions controlling firefly luciferase reporter plasmids were cotransfected into U2OS cells together with expression constructs of the aforementioned factors. Transfection of an empty expression vector (empty) served as negative control and differences in transfection efficiency were controlled by cotransfecting SV40 promoter driven renilla luciferase. Cells were cultured at 20% or 0.2% oxygen for 24 h before dual luciferase activities were determined. ( B ) Transient RNAi mediated knock down of HIF-1α fully abrogated hypoxic activation of the P2P by ETV4. U2OS cells were transiently transfected with siRNA oligonucleotides targeting HIF-1α (siHIF1α, right panel) or a control sequence having no human target (siControl, left panel). Reporter gene experiments using the P2P reporter construct with only wild-type HBS were performed as described in (A). The inset shows an immunoblot confirming the robust knock down of HIF-1α in U2OS cells. ( C ) ETV4 and HIF-1 synergism in hypoxic gene activation is not restricted to the P2P. A heterologous hypoxia responsive reporter gene containing two functional HBS from the human Transferrin hypoxia response element (pGL-TfHRE wt) was tested in luciferase reporter assays as described in (A). Mutation of both HBS (pGL-TfHRE mut) caused an abrogation of the signal as seen in (A). ( D and E ) Forced expression of ETV4 in U2OS cells upregulates endogenous PHD2 protein and transcript levels. ( D ) Whole cell lysates were prepared from cells exposed for 16 h to 20 or 0.2% oxygen and analyzed for HIF-1α, ETV4, PHD2 and β-actin levels by immunoblotting. ( E ) Total RNA was extracted of similarly treated cells and mRNA levels of PHD1, PHD2 and L28 were quantified by RT-qPCR. Data are shown in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (** P < 0.01, paired Student's t -test).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Activity Assay, Luciferase, Over Expression, Mutagenesis, Expressing, Construct, Transfection, Plasmid Preparation, Negative Control, Cell Culture, Knockdown, Activation Assay, Control, Sequencing, Western Blot, Functional Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. ( A ) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. ( B ) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O 2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). ( C ) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. ( D ) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). ( E ) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. ( F ) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. ( G ) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1 ) containing the HBS was determined by PCR followed by agarose gel electrophoresis.

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Construct, Activation Assay, Plasmid Preparation, Transfection, Incubation, Luciferase, Negative Control, Blocking Assay, Cotransfection, Expressing, Activity Assay, Knockdown, Western Blot, Control, Derivative Assay, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Genome-wide microarray expression analysis reveals a broad role for ETV4 in HIF mediated hypoxic gene regulation. ( A ) Efficient knock down of ETV4 in human PC3 prostate cancer cells. PC3 cells were stably transduced with lentiviral shRNA expression vectors encoding either a nontarget control (shNTC) or shETV4. Following 24 h of exposure to 20% O 2 or 0.2% O 2 , ETV4, HIF-1α, PHD2 and β-actin protein levels were analyzed by immunoblotting. ( B ) Total RNA was isolated from cultures treated as in (A) and mRNA levels of ETV4 and its target gene COX2 were determined by RT-qPCR. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments. ( C ) Venn diagram showing the number of transcripts regulated by either an at least twofold induction by hypoxia alone (red), an at least twofold reduction in normoxic cells by the knock down of ETV4 (green), or an at least twofold reduction in hypoxic cells by the knock down of ETV4 (blue), respectively. ( D ) Heatmap of the individual expression levels of the 47 transcripts that required ETV4 for efficient hypoxic induction. ( E ) Expression levels of four randomly chosen transcripts shown in (D) were confirmed by RT-qPCR as described for (B).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Genome Wide, Microarray, Expressing, Knockdown, Stable Transfection, Transduction, shRNA, Control, Western Blot, Isolation, Quantitative RT-PCR, Gene Expression

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Role of ETV4 in the regulation of established HIF target genes in vitro and in vivo . ( A ) Dot plots showing the correlation between transcripts in normoxic versus hypoxic control cells (left panel) or in hypoxic control versus hypoxic ETV4 knock down cells (right panel) as derived from the gene array data (grey dots). Red dots refer to internal controls and the blue dot shows ETV4 which is downregulated in shETV4 cells. Green dots indicate the positions of a predefined set of 61 well-established HIF target genes. ( B ) Heat map of the 61 HIF target genes ranked by the magnitude of ETV4 requirement for hypoxic induction according to differences in hypoxic expression levels with Δhyp = log 2 (shNTC_hypoxia) − log 2 (shETV4_hypoxia) and mean hypoxic expression levels centered to the mean of normoxic control cells. ( C ) Exemplary mRNA levels of HIF target genes which either require ETV4 for efficient hypoxic induction (PHD3 and CA9) or which remain unaffected by the ETV4 knock down (GLUT1 and PAI1). mRNA was quantified as described for B. ( D and E ) Correlation between ETV4 and established markers for tissue hypoxia in human breast cancer. ( D ) Independent specimens (spec.) of immunohistochemical evaluation of ETV4 expression in primary mammary carcinoma with high (upper panel) or low (lower panel) ETV4 expression levels. ( E ) Rank-order correlations (Spearman's rho) for ETV4 and PHD2 as well as known markers reflecting tissue hypoxia (HIF-1α, HIF-2α, PAI1, GLUT1 and CA9) are summarized in a cross table. The number of cases where both of the correlated markers could be assessed is displayed in parentheses. Asterisks indicate statistical significance with * P < 0.05 and ** P < 0.01.

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: In Vitro, In Vivo, Control, Knockdown, Derivative Assay, Expressing, Immunohistochemical staining

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 1. Epitope Mapping and Cellular Localization of Hec1 Monoclonal Antibody 9G3 (A) Peptides covering the sequence of human Hec1 were adsorbed onto nitrocellulose and immunoprobed with 9G3. As a control, HeLa extract was adsorbed onto the nitrocellulose at region H-12. Both the control spot and spot C-2 (amino acids 200–215) were positively identified. (B) Representation of the Ndc80 complex as predicted from previous publications (Wei et al., 2005; Ciferri et al., 2005). The asterisk marks the site on Hec1 where 9G3 binds. (C and D) Localization of 9G3 (green) and an antibody to Spc24 (red) in PtK1 cells (C) and HeLa cells (D). Linescans were carried out on sister kinet- ochore pairs from both HeLa cells (n = 40 pairs/3 cells) and PtK1 cells (n = 34 pairs/4 cells), and in all cases Hec1 localized exteriorly to Spc24 at kinetochores. (E) Western blot of whole-cell PtK1 extract with 9G3 as a probe. (F) Immunofluorescent image of a PtK1 cell injected with 9G3. To the right of each cell panel in (C), (D), and (F), a single kinetochore pair has been enlarged. The graphs represent the linescan data from the single kinetochore pair. Scale bars in (C), (D), and (F) = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Sequencing, Control, Western Blot, Injection

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 4. Loss of Kinetochore Oscillations and Plus-End MT Polymerization in Hec1 9G3-Injected Cells (A and B) Kinetochore behavior was analyzed by live-cell fluorescence timelapse imaging. Cells were injected with rhodamine-labeled tubulin and Alexa 488-conjugated CENP-F antibodies (A), or additionally with 9G3 (B). Images were acquired every 15 s. Selected planes are shown from the timelapse sequences (A and B, top). Selected kinetochore pairs are boxed and a time series of 12 images for each pair is shown below (A and B, bottom). Kinetochores from the control cell exhibited oscillatory behavior and periods of stretching and relaxation (example in A, bottom), whereas kinetochores from the 9G3-injected cell did not oscillate (example in B, bottom). (C and D) EB1-GFP-expressing PtK1 cells were injected with Texas Red dextran alone (C) or in combination with 9G3 (D). Images were acquired every 10 s. A region containing a kinetochore pair and the spindle poles was extracted from the timelapse sequence and shown to the right. The bright spots in extracted images are spindle poles. (E) A buffer-injected monopolar cell exhibits chromosome oscillations toward and away from the pole both prior to and after injection (top panel). In cells injected with 9G3, chromosomes stopped oscillating and moved poleward after injection (middle and bottom panels). In all panels, scale bars = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Injection, Imaging, Labeling, Control, Expressing, Sequencing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 6. Aurora B Kinase Phosphoryla- tion and Regulation of Hec1 (A) Left: Aurora B/INCENP790–856 in vitro kinase assay with Histone H3 as a control substrate (lanes 1 and 2) and Hec11–230 (lanes 3 and 4). Antibody 9G3 was added to the reaction mixtures in lanes 2 and 4. Right: Normalized quantification of radioactive phosphate for lanes 1–4. Molecular weight standards are indi- cated in kilodaltons. (B) PtK1 cells were transfected with WT-GFP- Hec1 (upper row) or mutant 6A-GFP-Hec1 (bot- tom three rows) for 40 hr prior to fixation for im- munofluoresence. Scale bar = 5 mm. (C) Cells transfected with WT-GFP-Hec1 and 6A-GFP-Hec1 were scored for chromosome alignment and assigned to one of three cate- goryies: chromosomes all aligned, chromo- somes mostly aligned (1–2 chromosomes off the metaphase plate), or chromosomes mostly unaligned (fewer than 3 aligned chromosomes) (n = 52 WT-GFP-Hec1 cells; n = 40 6A-GFP- Hec1 cells). (D) Cells were scored for merotelic kineto- chores. For WT-GFP-Hec1-expressing cells: n = 19 prometaphase cells, n = 31 metaphase/ near metaphase cells, and n = 11 anaphase cells. For 6A-GFP-Hec1-expressing cells: n = 47 prometaphase cells, n = 13 metaphase/ near metaphase cells, and n = 18 anaphase cells.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: In Vitro, Kinase Assay, Control, Molecular Weight, Transfection, Mutagenesis, Expressing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 7. Model for Hec1 Regulation of kMT Dynamics and Attachment (A) Mitotic spindle arrangement in a control cell (top) in which normal Aurora B phophorylation and dephosphorylation occur. kMT plus ends exhibit dynamic instability and undergo periods of attachment and detachment. Net polymerization at plus ends of kMTs is balanced by net depolymerization at minus ends. After addition of 9G3 (bottom), the N terminus of Hec1 can no longer be phosphorylated by Aurora B, and kMT detachment and dy- namic instability are suppressed. Minus-end depolymerization is not inhibited, and active depolymerases shorten kinetochore fibers. Hyper-stretch of centromeres arises from pulling forces exerted by the centrosome-associated minus-end organizing complexes as they maintain connection with the depolymerizing minus ends of the kinetochore fibers. (B) Regulation of kMT plus-end dynamic instability and attachment strength at three possible interfaces. Interface 1 is between the N terminus of Hec1 and the MT lattice; interface 2 is between the N terminus of Hec1 and a kinetochore-binding MAP, and interface 3 is between the MAP and the MT lattice (see text for details).

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Control, De-Phosphorylation Assay, Binding Assay