Journal:

Article Title: Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

doi: 10.1128/JVI.75.4.1672-1680.2001

Figure Lengend Snippet: Seven primary isolate nef genes and D. con nef were stably expressed in CEM cells. The function of these Nefs in CD4 and MHC class I downregulation and activation of PAK-2 was determined. The level of expression for each Nef was determined by Western blot analysis. (A) Two-color analysis for CD4 (PE) and MHC class I (FITC) cell surface expression in transduced CEM cells was determined by fluorescence-activated cell sorter (FACS) analysis. (Top left) CEM LXSN cells (negative control). (Top right) CEM LXSN cells (positive control). (B) Western blot analysis of Nef expression in extracts from transduced CEM cells. Control, CEM LXSN cell extracts. (C) Activation of p21-activated protein kinase-2 (Pak2) by Nef was assayed with extracts from transduced CEM cells. Control, CEM LXSN cell extracts. We have reported 233 Nef to be expressed at near the same level as SF2 Nef with a rabbit anti-Nef serum (36). The apparent reduced expression of 233 Nef in Fig. ​Fig.2B2B seems to result from a reduced immunoreactivity of 233 Nef to the sheep anti-SF2 Nef serum used for these studies. A similar observation was made for NefEE155QQ in reference 2.

Article Snippet: For analysis of cell surface CD4 and MHC class I levels, transduced CEM cells (5 × 10 5 ) were first incubated with mouse monoclonal anti-haplotype A1, A11, and A26 MHC class I antibody (One Lambda) for 20 min on ice, and then the cells were washed twice in 2 ml of ice-cold phosphate-buffered saline containing 5% calf serum and 0.1% NaN 3 .

Techniques: Stable Transfection, Activation Assay, Expressing, Western Blot, Fluorescence, Negative Control, Positive Control

Journal:

Article Title: Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

doi: 10.1128/JVI.75.4.1672-1680.2001

Figure Lengend Snippet: The effect of D90-1 derived mutations, A29V and A158V, on D.con Nef function in CEM cells was determined. Two-color analysis for CD4 (PE) and MHC class I (FITC) cell surface expression in transduced CEM cells was determined by FACS. (Top left) CEM LXSN cells (negative control). (Top right) CEM LXSN cells (positive control).

Article Snippet: For analysis of cell surface CD4 and MHC class I levels, transduced CEM cells (5 × 10 5 ) were first incubated with mouse monoclonal anti-haplotype A1, A11, and A26 MHC class I antibody (One Lambda) for 20 min on ice, and then the cells were washed twice in 2 ml of ice-cold phosphate-buffered saline containing 5% calf serum and 0.1% NaN 3 .

Techniques: Derivative Assay, Expressing, Negative Control, Positive Control

Journal:

Article Title: Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

doi: 10.1128/JVI.75.4.1672-1680.2001

Figure Lengend Snippet: The effects of mutations of S189R and F193I on D.con Nef function and R189S on D88-11 Nef function in CEM cells were determined. (A) Two-color analysis for CD4 (PE) and MHC class I (FITC) cell surface expression in transduced CEM cells was determined by FACS. (Top left) CEM LXSN cells (negative control). (Top right) CEM LXSN cells (positive control). (B) Western blot analysis of Nef expression in extracts from transduced CEM cells. Control, CEM LXSN cell extracts. (C) Activation of p21-activated protein kinase-2 (Pak2) by Nef was assayed with extracts from transduced CEM cells. Control, CEM LXSN cell extracts.

Article Snippet: For analysis of cell surface CD4 and MHC class I levels, transduced CEM cells (5 × 10 5 ) were first incubated with mouse monoclonal anti-haplotype A1, A11, and A26 MHC class I antibody (One Lambda) for 20 min on ice, and then the cells were washed twice in 2 ml of ice-cold phosphate-buffered saline containing 5% calf serum and 0.1% NaN 3 .

Techniques: Expressing, Negative Control, Positive Control, Western Blot, Activation Assay

Journal:

Article Title: Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

doi: 10.1128/JVI.75.4.1672-1680.2001

Figure Lengend Snippet: Summary of Nef point mutations and their associated phenotypes a

Article Snippet: For analysis of cell surface CD4 and MHC class I levels, transduced CEM cells (5 × 10 5 ) were first incubated with mouse monoclonal anti-haplotype A1, A11, and A26 MHC class I antibody (One Lambda) for 20 min on ice, and then the cells were washed twice in 2 ml of ice-cold phosphate-buffered saline containing 5% calf serum and 0.1% NaN 3 .

Techniques: Activation Assay

Journal: Genome Research

Article Title: Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution

doi: 10.1101/gr.265306.120

Figure Lengend Snippet: 5mC and 5hmC detection by enzymatic deamination method. (A) Principle of the methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5mC and 5hmC or with BGT alone (right) to protect 5hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C). The TET2 and BGT treatment results in the distinction of 5mC and 5hmC from C, whereas BGT treatment results in the distinction of 5hmC from C and 5mC. (B) Deaminated cytosines from unmethylated lambda genome display no observable sequence preference by APOBEC(5mC) deamination method. (C) False-positive methylation calling rate (nonconversion error rate) of each cytosine dinucleotides sequence context (CpA [CA], CpC [CC], CpG [CG], and CpT [CT]) estimated from the unmethylated lambda genome for the enzymatic deamination method (APOBEC(5mC), two WGBS performed in this study, i.e., BS kit 1 and BS kit 2, and six published WGBS experiments sampled from the ENCODE Project) (Supplemental Table S3). (D) Deamination of 5mC in the fully methylated XP12 genome results in no observable sequence preference by APOBEC(5hmC) enzymatic deamination method. (E) Distribution patterns of 5mCpG (blue) and 5hmCpG (red: 50 ng library; pink: 1 ng library) at various protein/DNA interaction sites. The absolute (smooth lines) and normalized (dotted lines) 5hmC and 5mC levels in the CpG context are depicted around TET1, RNA polymerase II, and CTCF binding sites, as well as at active transcription chromatin mark (H3K4me3), repressive chromatin mark (H3K27me3), active enhancer mark (H3K27ac), and general enhancer (H3K4me1 in the absence of H3K4me3) regions. Unbound sites that are randomly sampled from the reference genome server as a control. (F) Pearson's correlation between 5hmC measured using sequencing of enzymatically deaminated DNA (x-axis) versus LC-MS (y-axis) for various genomic DNA. There are two technical replicates of the APOBEC(5hmC) sequencing method for each sample. 5hmC levels are presented as 1000 percentage, and both axes use the log scale.

Article Snippet: Enzymatic deamination for 5hmC detection For 5hmC detection, 200 ng of mouse E14 genomic DNA was mixed with 10 ng unmethylated lambda DNA, 10 ng of T4gt phage DNA, and 1 ng of CpG methylated pUC19 DNA and then incubated with 20 U of BGT (NEB) in 1× NEBuffer 2 for 2 h at 37°C.

Techniques: Amplification, Sequencing, Methylation, Binding Assay, Control, Liquid Chromatography with Mass Spectroscopy