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:

Article Title: Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells

doi: 10.1111/j.1365-2249.2006.03115.x

Figure Lengend Snippet: Effect of anti-CD25 monoclonal antibody. (a) SKH1 mice were intraperitoneally administered 500 µg purified rat anti-CD25. The mice which received the antibodies were killed at 24 h (day 1), day 3, day 5 or day 7 after the administration of the antibody. Spleen and lymph nodes were then harvested. Single-cell suspensions were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 and phycoerythrin (PE)-labelled anti-mouse CD25 and analysed by fluorescence activated cell sorter (FACSCalibur). Results of injection of isotype rat Ig G are not shown. Day 0 indicates results of mice without anti-CD25 treatment. The numbers located above dot plots indicate the percentages of CD25+ cells in CD4+ cells. (b) To determine the expression of CD25 and forkhead boxp3 (Foxp3) in CD4+ cells after anti-CD25 antibody treatment, five groups of SKH1 mice were used. Group 1 received a single dose of 500 µg isotype IgG. Group 2 received a single dose of 500 µg anti-CD25. Group 3 received two doses of 500 µg anti-CD25, 5 days apart. Group 4 received three doses of 500 µg anti-CD25, 5 days apart. Group 5 received four doses of 500 µg anti-CD25, 5 days apart. At day 5 of the last injections, cells from the spleen and lymph node were stained by FITC-anti-CD4, PE-anti-Foxp3 and allophycocyanin (APC)-anti-CD25 and analysed by Cyan ADP flow cytometer. Data shown are results from lymph node cells in groups 1 and 2 after one anti-CD25 or IgG isotype injection. The numbers located above each dot plot in the left column indicate the percentages of CD25+ in Foxp3+ cells gated on CD4+ cells. The numbers located above each dot plot in the middle column indicate the percentages of CD25+ cells in CD4+ cells. The numbers located above each dot plot in the right column indicate the percentages of Foxp3+ cells in CD4+ cells. (c) Anti-CD25 inactivates Treg in a consistent manner: CD25+/Foxp3 (gated on CD4+), CD25+/CD4+ and Foxp3+/CD4+ cells in the lymph node were analysed after one, two, three and four cycles of anti-CD25 treatment, showing consistent inactivation of Treg, without physical depletion. Similar analyses for IgG1 isotype treatment obtained after one cycle treatment are shown on the far left for comparison. Similar results were obtained in the studies of the spleen cells (data not shown).

Article Snippet: The function of the secondary antibody was validated by using a sandwich method: the plate was coated with unlabelled monoclonal rat anti-mouse IgG1 (BD Biosciences), followed by purified standard mouse IgG1 lambda (BD Biosciences) in serial dilutions, then by biotin-labelled anti-mouse lambda chain (1,2,3) (BD Biosciences), followed by incubation of streptavidin–peroxidase polymer (Sigma) and then TMB substrate (Sigma).

Techniques: Purification, Staining, Fluorescence, Injection, Expressing, Flow Cytometry

Journal:

Article Title: Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells

doi: 10.1111/j.1365-2249.2006.03115.x

Figure Lengend Snippet: NC1-immunized SKH1 mice produce IgG antoantibodies specifically against recombinant NC1 protein. (a). Titratable enzyme-linked immunosorbent assay (ELISA) titres of autoantibodies. Three groups of female SKH1 mice were included. One group received rat anti-CD25 and mouse NC1 (n = 5, solid diamond), the second group received rat isotype and mouse NC1 (n = 5, solid square) and the third group received rat anti-CD25 and mouse albumin (n = 3, open circle). Mice sera were collected at the end of 5·5 months after the initial immunization and tested for the specific IgG against mouse NC1 by ELISA as described in Materials and methods. (b). Western blot analysis of NC1-specific autoantibodies. Two representative samples from each group are shown: mice treated with anti-CD25 and immunized with albumin (lanes 1 and 2); mice treated with anti-CD25 and immunized with NC1 (lanes 3 and 4); mice treated with rat IgG and immunized with NC1 (lanes 5 and 6). Each of the wells were loaded with 300 ng of recombinant mouse NC1 protein.

Article Snippet: The function of the secondary antibody was validated by using a sandwich method: the plate was coated with unlabelled monoclonal rat anti-mouse IgG1 (BD Biosciences), followed by purified standard mouse IgG1 lambda (BD Biosciences) in serial dilutions, then by biotin-labelled anti-mouse lambda chain (1,2,3) (BD Biosciences), followed by incubation of streptavidin–peroxidase polymer (Sigma) and then TMB substrate (Sigma).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot

Journal:

Article Title: Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells

doi: 10.1111/j.1365-2249.2006.03115.x

Figure Lengend Snippet: NC1-immunized SKH1 mice produce IgG autoantibodies which react specifically with skin basement membrane. Indirect immunofluorescence microscopy was used to examine the reactivity of autoreactive IgGs with the skin basement membrane zone component, as described in Materials and methods. Left and middle panels show IgG autoantibodies reacting with the skin basement membrane in mice immunized with NC1 in combinations of anti-CD25 or isotype control, respectively. The right panel shows the result of the serum from mouse treated with anti-CD25 and immunized with albumin. Images are representative for each group (original magnification ×40).

Article Snippet: The function of the secondary antibody was validated by using a sandwich method: the plate was coated with unlabelled monoclonal rat anti-mouse IgG1 (BD Biosciences), followed by purified standard mouse IgG1 lambda (BD Biosciences) in serial dilutions, then by biotin-labelled anti-mouse lambda chain (1,2,3) (BD Biosciences), followed by incubation of streptavidin–peroxidase polymer (Sigma) and then TMB substrate (Sigma).

Techniques: Immunofluorescence, Microscopy

Journal:

Article Title: Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells

doi: 10.1111/j.1365-2249.2006.03115.x

Figure Lengend Snippet: IgG class autoantibodies deposited to skin basement membrane in NC1-immunized mice. Direct immunofluorescence microscopy was performed to examine the deposit of IgG skin basement membrane zone, as described in Materials and methods. The left panel shows IgG deposits in mice treated with anti-CD25 and immunized with NC1, the middle panel shows IgG deposits in mice treated with rat IgG and immunized with NC1 and the right panel shows IgG deposits in mice treated with anti-CD25 and immunized with albumin. Images are representative for each group (original magnification ×40).

Article Snippet: The function of the secondary antibody was validated by using a sandwich method: the plate was coated with unlabelled monoclonal rat anti-mouse IgG1 (BD Biosciences), followed by purified standard mouse IgG1 lambda (BD Biosciences) in serial dilutions, then by biotin-labelled anti-mouse lambda chain (1,2,3) (BD Biosciences), followed by incubation of streptavidin–peroxidase polymer (Sigma) and then TMB substrate (Sigma).

Techniques: Immunofluorescence, Microscopy

Journal:

Article Title: Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells

doi: 10.1111/j.1365-2249.2006.03115.x

Figure Lengend Snippet: Anti-NC1 IgG subclasses and light chains [enzyme-linked immunosorbent assay (ELISA)].

Article Snippet: The function of the secondary antibody was validated by using a sandwich method: the plate was coated with unlabelled monoclonal rat anti-mouse IgG1 (BD Biosciences), followed by purified standard mouse IgG1 lambda (BD Biosciences) in serial dilutions, then by biotin-labelled anti-mouse lambda chain (1,2,3) (BD Biosciences), followed by incubation of streptavidin–peroxidase polymer (Sigma) and then TMB substrate (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay