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: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Differentially expressed genes (DEGs, P-FDR < 0.05) were identified by RNA-seq analysis in IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1 KO HepG2 cells after 72 hrs of induction by dox, comparing to controls (dox-conditions). Cutoff threshold (fold change > +/-1.5) is indicated by dotted lines. (A) Analysis of all DEGs (n=3251) detected for IFN-λ4-GFP or IFN-λ4-GFP-IFNLR1 KO cells. In blue - DEGs (n=2,735) specific to IFN-λ4-GFP and considered IFNLR1-dependent. In black - DEGs (n=145) shared between both groups and considered IFNLR1-independent. In orange - DEGs (n=371) specific to IFN-λ4-GFP-IFNLR1 KO . (B) DEGs of IFN-λ4-GFP analyzed in IFN-λ3-GFP transcriptome. In black - DEGs (n=1,506) shared in IFN-λ4-GFP and IFN-λ3-GFP and in blue - IFN-λ4-signature DEGs (n=1,229) detected in IFN-λ4-GFP but not in IFN-λ3-GFP producing cells. Additional details are provided in Fig. S3 and Table S6. (C , D) Cell cycle analysis of cells synchronized by 24 hrs of serum starvation, treated with or without dox (0.5 µg/ml) for 72 hrs and analyzed by flow cytometry after PI staining. The plot shows a representative picture and the percentage of cells in each phase of the cell cycle. All data are shown as mean± SEM from triplicate experiments. *, P < 0.05. (E , F) Bromodeoxyuridine (BRDU, %) incorporation indicating cell proliferation in HepG2 cells expressing IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1KO. Cells were cocultured with HepG2 cells labeled with Far Red proliferation dye, dox-induced for 72 hrs and treated with BRDU for 3 hrs before analysis. Gates show HepG2 cells exposed to IFN-λs (IFN-λ treated cells) and HepG2 expressing IFN-λs. P-values compare corresponding dox+ vs. dox-HepG2 cells, ** p<0.01, Student’s T-test. Graphs represent one of three independent experiments, each in biological triplicates.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: RNA Sequencing, Cell Cycle Assay, Flow Cytometry, Staining, BrdU Incorporation Assay, Expressing, Labeling

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: (A) Unsupervised clustering of activities of 54 regulons that were significantly and directionally enriched with HepG2-DEGs in the set of 885 of TCGA-LIHC regulons. The heatmap shows differences in activity scores (dES) for the IFN-λ4-enriched regulons organized by GSEA-2T results for the IFN-λ4-GFP, IFN-λ3-GFP and IFN-λ4-GFP-IFNLR1 KO DEG signatures. Cluster I: regulons with dES > 0 in IFN-λ4-GFP; cluster II: regulons with dES < 0 in IFN-λ4-GFP. Table S7 provides the regulon activity scores presented in Figure 2A. (B-G) GSEA-2T plots for IRF1 and IRF2, respectively, in DEG signatures for (B-C) IFN-λ3-GFP, ( D-E ) IFN-λ4-GFP, and ( F-G ) IFN-λ4-GFP-IFNLR1 KO . (H) Inhibition of proliferation in IFN-λ4-GFP HepG2 cells, for one of three independent experiments. Cells were treated with IRF1 siRNA for 24 hrs, labeled with Far Red proliferation dye and dox-induced at indicated concentrations for 72 hrs. Proliferation was assessed by flow cytometry with a graph representing the geometric mean expression of Far Red proliferation dye with higher values indicating reduced cell proliferation. P-values compare dox-treated control siRNA with dox-treated IRF1 siRNA. ** p<0.01, Student’s T-test. Below: Western blots showing IRF1 protein levels following siRNA knockdown.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Activity Assay, Inhibition, Labeling, Flow Cytometry, Expressing, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Representative confocal images of HepG2 cells transduced with a mammalian baculovirus delivery system (BacMam) of GFP-tagged proteins targeting specific organelles - lysosomes, Golgi, early and late endosomes. After transduction for 6 hrs, cells were transiently transfected with Halo-tagged constructs for IFN-λ4 or control for indicated times, stained with cell-permeant Halo-tag ligand TMR (red), and imaged. (A) Confocal images showing IFN-λ4 accumulation in lysosomes but not in early endosomes. (B) Late endosomal trafficking of IFN-λ4, with the inset showing larger magnification. (C) Unfolded protein response (UPR) is represented by lysosomal enlargement after protein accumulation. (D) Live images of IFN-λ4-expressing HepG2 cells undergoing apoptosis, characterized by membrane blebbing and cell death. Images were scanned every minute for 12 hrs. Scale bars – 10 um. (E) Apoptosis detection with ApoTox-Glo assays in corresponding untreated and dox-induced cells for indicated time points. RLU, relative luminescence units. (F) Graph showing counts from colony formation assay for HepG2 cells expressing IFN-λ4 or IFNLR1 KO grown in 6-well plates with or without dox for 13 days. Cell colonies were stained with crystal violet and manually counted. The graph represents the number of colonies as a percentage of initial plated counts. (G,H) mRNA (G) and protein levels ( H ) of DDIT3 after siRNA knockdown tested by qRT-PCR and Western blot assays, respectively. (I-J) Apoptosis (I) and cell viability (J) assays were performed after siRNA knockdown of DDIT3 in dox-induced IFN-λ4-GFP cells. * p<0.05, ** p< 0.01, *** p<0.001.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Transduction, Transfection, Construct, Control, Staining, Expressing, Membrane, Colony Assay, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Cell reports

Article Title: Mapping the Lineage Relationship between CXCR5 + and CXCR5 − CD4 + T Cells in HIV-Infected Human Lymph Nodes

doi: 10.1016/j.celrep.2019.08.037

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-human Lambda, Unconjugated, polyclonal , Southern Biotech , Cat# 2070-01; RRID:AB_2795749.

Techniques: Recombinant, Staining, Labeling, Isolation, Sequencing, Reverse Transcription, Software

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars). The allotypes shown are those common to both beadsets.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of MA2.1 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Alignment of HLA class I allotypes showing selected residues in the α1 and α2 domains. Residues from allotypes that form the epitope recognized by MA2.1 are shaded in grey. (C) Space-filling model of the binding surface of HLA-A*02 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by MA2.1. Residues 62–65 are critical for formation of the epitope recognized by MA2.1 and are highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of PA2.1 (1μg/ml) and BB7.2 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Binding of PA2.1 (50μg/ml) and BB7.2 (50μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (C) Alignment of HLA class I allotypes showing selected residues in the α2 domain. Residues from allotypes that form the epitope recognized by PA2.1 and BB7.2 are shaded in grey. (D) Space-filling model of HLA-A*02 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by PA2.1 and BB7.2. Tryptophan at position 107 is considered critical for formation of the epitope recognized by PA2.1 and BB7.2 and is highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of BB7.1 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Alignment of HLA class I allotypes showing selected residues in the α1 and α2 domains. Residues from allotypes that form the epitope recognized by BB7.1 are shaded in grey. (C) Space-filling model of the binding surface of HLA-B*07 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by BB7.1. Residues 63–71 in the a1 domain and position 131 in the α2 domain are critical for formation of the epitope recognized by BB7.1 and are highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: AIDS Research and Therapy

Article Title: The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients

doi: 10.1186/s12981-017-0158-7

Figure Lengend Snippet: Nucleotide sequences of the primers used for real-time PCR

Article Snippet: Antibodies (Abs) used in this study were as follows: mouse phycoerythin (PE)-conjugated anti-human IFN-alpha/beta R2 Ab (clone MMHAR-2, R&D Systems), mouse PE-conjugated anti-human IFN-lambdas R1 Ab (clone 601106, R&D Systems), mouse PE-conjugated anti-human IFN-gamma Receptor 1 (clone GIR-208, Thermo Fisher Scientific), Mouse eFluor ® 450-conjugated anti-human STAT1 (KIKSI0803, Thermo Fisher Scientific), Rabbit FITC anti-Human STAT2 (Thermo Fisher Scientific), Mouse PE-Cyanine7-conjugated anti-human STAT3 (clone LUVNKLA, Thermo Fisher Scientific), mouse APC-conjugated anti-human STAT4 (clone 4LURPLE, Thermo Fisher Scientific), mouse FITC-conjugated anti-human STAT5 (clone SRBCZX, Thermo Fisher Scientific), mouse PerCP-eFluor ® 710-conjugated anti-human STAT6 (clone CHI2S4N, Thermo Fisher Scientific).

Techniques: Sequencing

Journal: AIDS Research and Therapy

Article Title: The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients

doi: 10.1186/s12981-017-0158-7

Figure Lengend Snippet: Correlation between the CD4 + T cells and mRNA levels of IFN-alpha receptor ( a ), IFN-gamma receptor ( c ), and IFN-lambdas receptor ( e ). Correlation between the viral loads and mRNA levels of IFN-alpha receptor ( b ), IFN-gamma receptor ( d ), and IFN-lambdas receptor ( f ). The results were performed Spearman’s rank correlation, where coefficients “r” and corresponding p values are indicated on each panel

Article Snippet: Antibodies (Abs) used in this study were as follows: mouse phycoerythin (PE)-conjugated anti-human IFN-alpha/beta R2 Ab (clone MMHAR-2, R&D Systems), mouse PE-conjugated anti-human IFN-lambdas R1 Ab (clone 601106, R&D Systems), mouse PE-conjugated anti-human IFN-gamma Receptor 1 (clone GIR-208, Thermo Fisher Scientific), Mouse eFluor ® 450-conjugated anti-human STAT1 (KIKSI0803, Thermo Fisher Scientific), Rabbit FITC anti-Human STAT2 (Thermo Fisher Scientific), Mouse PE-Cyanine7-conjugated anti-human STAT3 (clone LUVNKLA, Thermo Fisher Scientific), mouse APC-conjugated anti-human STAT4 (clone 4LURPLE, Thermo Fisher Scientific), mouse FITC-conjugated anti-human STAT5 (clone SRBCZX, Thermo Fisher Scientific), mouse PerCP-eFluor ® 710-conjugated anti-human STAT6 (clone CHI2S4N, Thermo Fisher Scientific).

Techniques: