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