Journal: Cells

Article Title: Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

doi: 10.3390/cells8121590

Figure Lengend Snippet: Construction and characterization of fluorescent transporter associated with antigen processing (TAP)-green fluorescent protein (GFP) variants. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts (T1KO, T2KO) were stably reconstituted with fluorescent TAP1 or TAP2 constructs using lentivirus vectors and cell sorting. ( A ) Schematic representation of TAP-GFP constructs. Secondary structures of linkers flanked by ten amino acid residues of fused proteins were determined by the Geneious software; α-helices are depicted in pink, coiled regions in gray, β-strands in yellow, and turns in blue. ( B ) Representative histograms of TAP-GFP fluorescence intensity. ( C ) Comparative TAP-GFP analysis by flow cytometry. The mean fluorescence intensity of three independent measurements is represented as bars with standard deviations. The statistical significance was assessed by t -test; p ≤ 0.001. ( D – F ) Expression of TAP-GFP variants in stable cell lines was determined by SDS-PAGE and immunoblotting using: ( D ) anti-GFP monoclonal antibody (Mab) ( E ) anti-TAP2 MAb ( F ) anti-TAP1 MAb. β-actin was used as a loading control. Abbreviations: T2-C: TAP2-C-GFP (TAP2 with the C-terminal GFP, random linker); T2-N: TAP2-N-GFP (TAP2 with the N-terminal GFP, random linker); T1-C: TAP1-C-GFP (TAP1 with the C-terminal GFP, random linker); T1-HC: TAP1-HC-GFP (TAP1 with the C-terminal GFP, helical linker); T1-N: TAP1-N-GFP (TAP1 with the N-terminal GFP, random linker); T1-HN: TAP1-HN-GFP (TAP1 with the N-terminal GFP, helical linker).

Article Snippet: Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [ ] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [ ] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology).

Techniques: CRISPR, Stable Transfection, Construct, FACS, Software, Fluorescence, Flow Cytometry, Expressing, SDS Page, Western Blot

Journal: Cells

Article Title: Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

doi: 10.3390/cells8121590

Figure Lengend Snippet: TAP-GFP forms a functional transporter. MJS cells with CRISPR/Cas9 TAP1 or TAP2 knockouts (T1KO or T2KO) were stably reconstituted with wild-type or fluorescent TAP1 or TAP2 variants. Surface expression of major histocompatibility complex class I (MHC I) was assessed by flow cytometry using specific antibodies (W6/32). MHC I expression on MJS cells with TAP reconstitution is presented as the percentage of MHC I mean fluorescence intensity on MJS cells (set as 100%). The analysis was performed in triplicates. The statistical significance of differences between MHC I on MJS cell with TAP reconstitution and MJS wild-type (wt) cells was estimated by t -test; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ns: not significant.

Article Snippet: Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [ ] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [ ] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology).

Techniques: Functional Assay, CRISPR, Stable Transfection, Expressing, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

doi: 10.3390/cells8121590

Figure Lengend Snippet: TAP-GFP variants differ in their sensitivity to UL49.5-mediated inhibition and degradation. MJS cells with fluorescent TAP1 or TAP2 variants were transduced with a retrovirus encoding bovine herpesvirus 1 (BoHV-1) UL49.5. ( A ) Surface expression of MHC I was assessed by flow cytometry using specific antibodies (W6/32). MHC I expression is presented as the percentage of mean fluorescence intensity; fluorescence of parental cells without UL49.5 was set as 100%. The analysis was performed in triplicates. The statistical significance of differences between MJS TAP-GFP and MJS TAP-GFP UL49.5 cell lines was estimated by t -test; ** p ≤ 0.001 * p ≤ 0.005. ( B ) GFP mean fluorescence intensity is presented as the percentage of GFP fluorescence of parental cells (set as 100%). The analysis was performed in triplicates. The statistical significance of differences between MJS TAP-GFP and MJS TAP-GFP UL49.5 cell lines was estimated by t -test; ** p ≤ 0.001 * p ≤ 0.005. ( C ) The effect of UL49.5 on GFP level in MJS TAP-GFP cells was assessed by flow cytometry. ( D , E ) Degradation of TAP-GFP variants in the presence of BoHV-1 UL49.5 in stable cell lines was determined by SDS-PAGE and immunoblotting using: anti-GFP, anti-TAP1, anti-TAP2, or anti-UL49.5 antibodies. β-catenin was used as a loading control.

Article Snippet: Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [ ] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [ ] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology).

Techniques: Inhibition, Transduction, Expressing, Flow Cytometry, Fluorescence, Stable Transfection, SDS Page, Western Blot

Journal: Cells

Article Title: Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

doi: 10.3390/cells8121590

Figure Lengend Snippet: TAP1-GFP interacts with UL49.5 and the peptide-loading complex. TAP1-HN-GFP (T1-HN) was immunoprecipitated by GFP-Trap from lysates of MJS cells expressing TAP1-HN-GFP and UL49.5 or wt MJS with UL49.5 only. Co-precipitating proteins were analyzed by SDS-PAGE and immunoblotting using antibodies against GFP, TAP2, ERp57, MHC I HC, and UL49.5. Right panel: cell lysates were loaded on SDS-PAGE directly and analyzed by immunoblotting.

Article Snippet: Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [ ] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [ ] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Expressing, SDS Page, Western Blot

Journal: Cells

Article Title: Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

doi: 10.3390/cells8121590

Figure Lengend Snippet: BoHV-1 infection results in TAP2-GFP degradation. MJS TAP2-N-GFP cells were infected with BoHV-1 at a multiplicity of infection (moi) = 10. Twenty-four hours post-infection, cells were collected and analyzed. ( A ) TAP2-GFP fluorescence was assessed by flow cytometry; histograms from a representative analysis are shown, and ( B ) depicted as the percentage of fluorescence in mock-infected MJS TAP2-N-GFP cells (set as 100%). The analysis was performed in triplicates. The statistical significance was assessed by t -test; p ≤ 0.0005 ( C ) TAP2-GFP degradation was determined by SDS-PAGE and immunoblotting using anti-GFP, anti-TAP1, or anti-UL49.5 antibodies; β-actin was used as a loading control. ( D ) The relative amount of TAP2-GFP detected by immunoblotting was normalized to β-actin.

Article Snippet: Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [ ] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [ ] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology).

Techniques: Infection, Fluorescence, Flow Cytometry, SDS Page, Western Blot

Journal: Cells

Article Title: Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

doi: 10.3390/cells8121590

Figure Lengend Snippet: UL49.5 induced TAP-GFP degradation is p97-dependent. MJS TAP2-N-GFP UL49.5 cells were treated with p97 inhibitor NMS-873 (NMS) at 2 µM concentration for 24 hours. ( A ) Flow cytometry analysis of GFP fluorescence in NMS-treated and control (DMSO-treated) cells. ( B ) Relative GFP fluorescence in NMS-treated cells calculated as a percentage of GFP fluorescence in the control cells. The analysis was performed in triplicates. ( C ) The level of TAP-GFP in the presence of p97 inhibitor was determined by SDS-PAGE and immunoblotting using anti-GFP, anti-TAP1 or anti-UL49.5 antibodies; β-actin was used as a loading control. ( D ) Surface expression of MHC I assessed by flow cytometry using specific antibodies (W6/32).

Article Snippet: Antibodies used for immunoblotting: mouse anti-TAP1 monoclonal antibody MAb 143.5 (kindly provided by R. Tampé, Institute of Biochemistry, The Johann Wolfgang Goethe University, Frankfurt, Germany); mouse anti-TAP2 MAb 435.3 (a kind gift from P. van Endert, INSERM U25, Institute Necker, Paris, France); rabbit anti-TAP1 (Enzo Life Sciences, Farmingdale, NY, USA); rat anti-GFP 3H9 (Chromotek, Planegg, Germany); mouse anti-myc tag 9B11 (Cell Signaling, Danvers, MA, USA); rabbit anti-β-actin (Novus Biologicals, Centennial, CO, USA); rabbit anti-β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit antibodies (H11) against a synthetic peptide derived from the N-terminal domain of BoHV-1 UL49.5 [ ] and mouse anti-OctA (FLAG) G-8 (Santa Cruz Biotechnology); and mouse anti-HC10 [ ] and rabbit anti-ERp57 H-220 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Flow Cytometry, Fluorescence, SDS Page, Western Blot, Expressing

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: Immunohistochemical stainings of TAP1 in CRC specimens. Representative light microscopic images (x20 objective magnification) of tumor specimens with low (left) and high (right) TAP1 expression.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Immunohistochemical staining, Expressing

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: TAP1 expression in relation to clinicopathological characteristics in CRC.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Expressing, Significance Assay

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: TAP1 expression in relation to molecular characteristics in CRC.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Expressing

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: The correlation of expression of TAP1 with immune cell markers.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Expressing, Significance Assay

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: TAP1 expression in relation to expression of MHC class I (HLA-A, B and C) and TAP2.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Expressing

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: Cancer-specific survival in CRC patients. Kaplan-Meier plots of cases scored for TAP1 expression in the tumor front and tumor center: IRS ≤ 6, low expression; IRS > 6, high expression. Log-rank tests were used to calculate P-values.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Expressing

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: Cox regression analyses of TAP1 expression in predicting survival of CRC patients.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Expressing, Significance Assay

Journal: Oncoimmunology

Article Title: TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

doi: 10.1080/2162402X.2017.1356143

Figure Lengend Snippet: TAP1 methylation and expression in CRC. (A) A schematic illustration of the TAP1 gene. Methylation of putative CpG regions correlated with TAP1 expression, as identified from the TCGA data set, are highlighted by boxes. (B) Percent methylation of specific CpG-sites (a-c) (described in Supplementary Table 3) as analyzed by pyrosequencing and illustrated by Boxplots. Outlier values (o) and far-out values (*) are indicated. The analyses include patients with high (H, IRS > 6, n = 10) and low (L, IRS ≤ 6, n = 12) expression of TAP.

Article Snippet: The anti-TAP1 antibody (11114–1-AP; Nordic Biosite) and the anti-TAP2 antibody (K0137–3; MBL) were used at a dilution of 1:50, and the anti-HLA class I ABC antibody (ab70328; Abcam) at a dilution of 1/400 on an automated Ventana Benchmark Ultra staining machine with the ultraVIEW DAB Detection Kit for visualization (Ventana Medical Systems, Inc.).

Techniques: Methylation, Expressing