Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: Polyubiquitinated p53 species detected in U2OS cells titrated with LMP1 . Western blot detection of transfected LMP1, endogenous p53 protein levels and detection of ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP1 (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increased polyubiquitinated p53 protein was seen as high molecular weight aggregates in LMP1 expressing cells.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control, High Molecular Weight, Expressing

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: PI3K-Akt inhibitor LY294002 does not restore p53 protein levels . (A) Western blot analysis of the effects of PI3K inhibitor LY294002 on the endogenous p53 protein levels in U2OS cells. The cells were treated overnight, with 25 μM LY294002 or vehicle alone (DMSO). Treatment by LY294002 did not rescue the reduction of p53 protein levels by LMP1. (B) Western blot analysis of the effects of PI3K inhibitor LY294002 on the transfected p53 protein levels in CNE1-LMP1 stable cell line. The cells were treated overnight with 25 μM LY294002 or vehicle alone (DMSO). β-actin was served as a loading control in both the experiments. Representative blots were quantitated using Image J. Treatment with LY294002 did not rescue the reduction of p53 protein levels by LMP1.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Transfection, Stable Transfection, Control

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: LMP1, but not LMP2A or EBNA1, reduces p53 protein levels . (A) Western blot analysis of the effects of LMP1, LMP2A and EBNA1 on the expression levels of endogenous p53 protein in U2OS cells. Transfection of LMP1 reduced the level of p53 protein. (B) Co-transfection of LMP1 abolished p53 protein level in HONE1 NPC cells transiently transfected with p53. A slight reduction in the exogenous p53 protein level in LMP2A co-transfected in HONE1 NPC cells setting but not in U2OS cells in (A). ( C ) EBV-negative HONE1 and EBV-positive HONE Akata (HA) cells were transfected with p53 construct. p53 protein levels were determined in the Actinomycin-D induced and uninduced state. EBV-positive HA NPC cells have lower levels of p53 protein in comparison with EBV-negative HONE NPC cells. β-actin was served as a loading control in all the three experiments. Representative blots were quantitated using Image J.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Expressing, Transfection, Cotransfection, Construct, Comparison, Control

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: Only monoubiquitinated p53 species detected in U2OS titrated with LMP2A . Western blot detection of transfected LMP2A-HA, endogenous p53 protein levels and ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP2A-HA (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. No polyubiquitinated p53 protein was seen in LMP2A expressing cells.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control, Expressing

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: Dominant-negative Akt (DN-Akt) does not restore p53 protein levels . Western blot detection of LMP1, Dominant-negative Akt (DN-Akt) and p53 protein levels in U2OS cells transfected with fixed amount of LMP1 (0.5 μg) and titrated with DN-Akt (0.05 - 4.0 μg). β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increasing amounts of DN-Akt (detected by anti-Akt antibodies) did not rescue reduction of p53 protein levels by LMP1.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Dominant Negative Mutation, Western Blot, Transfection, Control

Journal: Frontiers in Immunology

Article Title: T cell-mediated immune surveillance conferred by latent Epstein-Barr virus genes suppresses a broad spectrum of tumor formation through NKG2D-NKG2DL interactions

doi: 10.3389/fimmu.2025.1597731

Figure Lengend Snippet: Characterization of germinal center B cell-specific LMP1/2A-expressing mice. (A) Targeting strategy for conditional expression of LMP1 and LMP2A in germinal center (GC) B cells. LMP1, LMP2A, and EYFP reporter genes were targeted into the Rosa26 locus with the neomycin resistance stop cassette enabling constitutive expression of those genes after Cre-mediated excision of stop cassette (R26-LMP1/2A(iresEYFP) flSTOP mouse) and crossed with GC B cell-specific Cre strain, Cγ1-Cre mice. SA, splice acceptor. (B) Workflow for isolating and culturing LMP1/2A + B cells from GCB-LMP1/2A mice ( left ). Morphological changes of B cells during culture, showing increased cell size and aggregation into large clumps ( right ). Scale bar, 100 μm. (C) Growth curves of in vitro cultured B cells from control and GCB-LMP1/2A mice (n=3). Statistical significance tested using two-way ANOVA with Bonferroni’s multiple comparisons test; ** p < 0.01; **** p < 0.0001. (D) FACS analysis of in vitro cultured B cells. Proliferating cells expressed Fas, confirming their GC B cell identity. EYFP was used as a reporter for LMP1/2A expression. (E, F) Percentage and cell count of GC B cells (E) and CD8 + T EM cells (F) in the spleens of GCB-LMP1/2A mice compared to control mice (n=6). Statistical significance tested using an unpaired two-tailed Student’s t-test; *** p < 0.001.

Article Snippet: The following antibodies were used: mouse anti-EBV LMP1 (S12,Sigma-Aldrich), rat anti-EBV LMP2A (14B7, Santa Cruz Biotechnology), rabbit anti-β-actin (Proteintech), HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich), HRP-conjugated donkey anti-rat IgG (Jackson ImmunoResearch), and HRP-conjugated goat anti-rabbit IgG (Proteintech).

Techniques: Expressing, In Vitro, Cell Culture, Control, Cell Counting, Two Tailed Test

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: Polyubiquitinated p53 species detected in U2OS cells titrated with LMP1 . Western blot detection of transfected LMP1, endogenous p53 protein levels and detection of ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP1 (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increased polyubiquitinated p53 protein was seen as high molecular weight aggregates in LMP1 expressing cells.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control, High Molecular Weight, Expressing

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: PI3K-Akt inhibitor LY294002 does not restore p53 protein levels . (A) Western blot analysis of the effects of PI3K inhibitor LY294002 on the endogenous p53 protein levels in U2OS cells. The cells were treated overnight, with 25 μM LY294002 or vehicle alone (DMSO). Treatment by LY294002 did not rescue the reduction of p53 protein levels by LMP1. (B) Western blot analysis of the effects of PI3K inhibitor LY294002 on the transfected p53 protein levels in CNE1-LMP1 stable cell line. The cells were treated overnight with 25 μM LY294002 or vehicle alone (DMSO). β-actin was served as a loading control in both the experiments. Representative blots were quantitated using Image J. Treatment with LY294002 did not rescue the reduction of p53 protein levels by LMP1.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Transfection, Stable Transfection, Control

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: LMP1, but not LMP2A or EBNA1, reduces p53 protein levels . (A) Western blot analysis of the effects of LMP1, LMP2A and EBNA1 on the expression levels of endogenous p53 protein in U2OS cells. Transfection of LMP1 reduced the level of p53 protein. (B) Co-transfection of LMP1 abolished p53 protein level in HONE1 NPC cells transiently transfected with p53. A slight reduction in the exogenous p53 protein level in LMP2A co-transfected in HONE1 NPC cells setting but not in U2OS cells in (A). ( C ) EBV-negative HONE1 and EBV-positive HONE Akata (HA) cells were transfected with p53 construct. p53 protein levels were determined in the Actinomycin-D induced and uninduced state. EBV-positive HA NPC cells have lower levels of p53 protein in comparison with EBV-negative HONE NPC cells. β-actin was served as a loading control in all the three experiments. Representative blots were quantitated using Image J.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Expressing, Transfection, Cotransfection, Construct, Comparison, Control

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: Only monoubiquitinated p53 species detected in U2OS titrated with LMP2A . Western blot detection of transfected LMP2A-HA, endogenous p53 protein levels and ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP2A-HA (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. No polyubiquitinated p53 protein was seen in LMP2A expressing cells.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control, Expressing

Journal: BMC Research Notes

Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

doi: 10.1186/1756-0500-4-551

Figure Lengend Snippet: Dominant-negative Akt (DN-Akt) does not restore p53 protein levels . Western blot detection of LMP1, Dominant-negative Akt (DN-Akt) and p53 protein levels in U2OS cells transfected with fixed amount of LMP1 (0.5 μg) and titrated with DN-Akt (0.05 - 4.0 μg). β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increasing amounts of DN-Akt (detected by anti-Akt antibodies) did not rescue reduction of p53 protein levels by LMP1.

Article Snippet: Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

Techniques: Dominant Negative Mutation, Western Blot, Transfection, Control