registration of intensity profiles using dapi stained-chromatin as reference  (MathWorks Inc)

Journal: Nucleic Acids Research

Article Title: Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

doi: 10.1093/nar/gkab886

Figure Lengend Snippet: Binding of human LEDGF/p75 and HIV-1 IN to chromosomes spreads. Either recombinant purified LEDGF/p75 or HIV-1 IN (4nM) were incubated alone (respectively A and B ) or together ( C ) with chromosomes spreads under the conditions described in materials and methods section. Monitoring of their interaction with the chromatin was performed by immunofluorescence with the corresponding antibody coupled to ALEXA 594 (green signal) and DAPI staining (red signal). The intensity of total IF signal has been quantified in each conditions using ImageJ software and the IN binding without or with LEDGF/p75 were reported in ( D ) as mean from the quantification of 7–11 chromosomes in each condition ± standard deviation (SD), P -value was calculated by student test. Scale bar = 10 μM.

Article Snippet: Both registration of intensity profiles using DAPI stained-chromatin as reference (see S2), and correlation between the DAPI stained-chromatin and the protein distribution was performed using MATLAB.

Techniques: Binding Assay, Recombinant, Purification, Incubation, Immunofluorescence, Staining, Software, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

doi: 10.1093/nar/gkab886

Figure Lengend Snippet: Correlation analysis between the protein distributions and chromatin features. Distributions of IN, LEDGF/p75 and IN-LEDGF/p75 were compared to DAPI staining and histones distribution. The mean and standard deviation of the pearson correlation calculation (>0 for a correlation, =0 for no correlation and < 0 for a negative correlation) were performed between all paired protein distribution and DAPI/histone curves. The p-value (Wilcoxon rank sum test) at each position and the percentage of pairs with P < 0.01 was also calculated and were reported as *** if > 0.80 and ** if > 0.40).

Article Snippet: Both registration of intensity profiles using DAPI stained-chromatin as reference (see S2), and correlation between the DAPI stained-chromatin and the protein distribution was performed using MATLAB.

Techniques: Staining, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75

doi: 10.1093/nar/gkab886

Figure Lengend Snippet: Analysis of the chromatin binding property of HIV-1 intasome. HIV-1 intasome was assembled using IN, LEDGF/p75 and the viral short DNA sequence corresponding to the viral end tagged with FITC. The intasome was purified by size exclusion chromatohraphy (see S8). After verification of their functionality in in vitro concerted integration assays the intasome (4nM) were incubated with chromosomes spreads. Interaction profile was determine by FITC-epifluorescence acquisitions ( A ). The distribution profile onto chromosome 1 was compared determined as reported in materials and methods section (an example of chromosome 1 is reported in ( B ), see the profile of chromosome 2 and 3 in S9). The data are reported as means from the quantification of eight to nine chromosomes ± standard deviation (SD). Scale bar = 10 μM. Correlation between the distributions of the intasome and the distribution of IN alone, LEDGF/p75 alone or IN-LEDGF/p75 complex ( C ) as well as DAPI staining, H3/H4, H3K36me3, K3K27me3 and H3K27ac ( D ) was compared. The mean and standard deviation of the Pearson correlation calculation (>0 for a positive correlation, =0 for no correlation and <0 for negative correlation) were performed between all paired protein distribution and DAPI/histone curves. The P -value (Wilcoxon rank sum test) at each position and the percentage of pairs with P < 0.01 was also calculated and were reported as *** if > 0.80 and ** if > 0.40).

Article Snippet: Both registration of intensity profiles using DAPI stained-chromatin as reference (see S2), and correlation between the DAPI stained-chromatin and the protein distribution was performed using MATLAB.

Techniques: Binding Assay, Sequencing, Purification, In Vitro, Incubation, Standard Deviation, Staining