Journal: Cell

Article Title: Selective activation of PFKL suppresses the phagocytic oxidative burst

doi: 10.1016/j.cell.2021.07.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-phosphoMEK1/2 (pMEK) 4169 , Cell Signaling Tech. , 9154S; RRID:AB_2138017.

Techniques: Virus, Cloning, Recombinant, CellTox Assay, Cytotoxicity Assay, Cytokine Assay, Isolation, Activity Assay, Screening Assay, Mass Spectrometry, Software

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: GST-TAF4 (lanes 3 and 6) or GST (lanes 2, 5) was added to the lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate (lanes 1–3) or from E. coli BL21(DE3)(pET-Rta) (lanes 4–6). Proteins bound to GST-TAF4 were pulled down by glutathione-Sepharose beads and analyzed by immunoblotting (IB) with anti-Rta antibody. Lanes 1 and 4 were loaded with 5% of cell lysate. GST or GST-TAF4 bound to glutathione-Sepharose beads were analyzed by immunoblotting (IB) with anti-GST antibody (lanes 7, 8).

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: Interaction between TAF4 and Rta was examined using P3HR1 cells that were treated with DMSO (lanes 5–8; 13–16) or TPA and sodium butyrate (lanes 1–4; 9–12) for 24 hr. Proteins in the cell lysate were immunoprecipitated with anti-Rta (lanes 4, 8, 11, 15), anti-TAF4 (lanes 3, 7, 12, 16) or anti-IgG (lanes 2, 6, 10, 14) antibody. Immunoprecipitated proteins were detected by immunoblotting with anti-Rta (lanes 1–8) or anti-TAF4 (lanes 9–16) antibody. Lanes 1, 5, 9 and 13 were loaded with 5% of the cell lysate.

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Transfection, Incubation, Laser-Scanning Microscopy, Staining

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Western Blot, Transfection

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: (A) Biotin-labeled double-stranded F23 probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate for 52 hr. Mutant probes mF23 that contains mutated sequences (asterisk) was used as negative controls. Proteins bound to the probes were captured by the streptavidin magnetic beads and detected by immunoblotting analysis using anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA affinity precipitation assay. (B) P3HR1 cells were treated with TPA and sodium butyrate for 52 hr. Formaldehyde-fixed DNA-protein complex was immunoprecipitated using anti-TAF4 or anti-Rta antibody. The reaction with added anti-IgG antibody was used as a negative control. The binding of TAF4 and Rta to the BcLF1 promoter was investigated by qPCR. Error bar represents standard error. (C) 293T cells were cotransfected with pCMV-R and pGL2-F23 or a control vector pGL2-basic in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (shTAF4) (empty column). Luciferase activity was detected at 48 hr after transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate. (D) The effect of TAF4 shRNAs was examined by immunoblotting with anti-TAF4 and anti-α-tubulin antibodies. The p value from each experiment was analyzed statistically with the Student's t- test method.

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Labeling, Mutagenesis, Magnetic Beads, Western Blot, Affinity Precipitation, Immunoprecipitation, Negative Control, Binding Assay, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Transfection

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: (A) The TR-L1 reporter plasmid and sequences of biotin-labeled probes, TR-L1 and mTR-L1. The probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins bound to the probes were captured by streptavidin magnetic beads and detected by immunoblotting analysis using anti-Sp1, anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 hr (lytic) or treated with DMSO (latent) were fixed with formaldehyde and the DNA-protein complexes were immunoprecipitated using anti-Sp1, anti-TAF4 and anti-Rta antibodies. The binding of Sp1, TAF4 and Rta to the TR-L1 and BALF5 promoters was investigated by qPCR. Error bar represents standard error. (C) 293T cells were cotransfected with pCMV-R and reporter plasmids, including pTRL1-luc and pBALF5-luc in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (empty column). Luciferase activities were monitored at 48 hr after transfection. Each transfection experiment was performed at lease three times, and each sample in the experiment was prepared in duplicate. (D) A similar experiment in (C) was performed using pBMRF1-luc and pBMRF1-mRRE-luc. (E) The effect of TAF4 shRNAs on the expression of TAF4 was examined by immunoblotting using anti-TAF4 and anti-α-tubulin antibodies. The p value from each experiment was analyzed statistically with the Student's t- test method. Luc: luciferase gene.

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Plasmid Preparation, Labeling, Magnetic Beads, Western Blot, Affinity Precipitation, Immunoprecipitation, Binding Assay, shRNA, Luciferase, Transfection, Expressing

Journal: PLoS ONE

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

doi: 10.1371/journal.pone.0054075

Figure Lengend Snippet: (A) An hAR reporter plasmid and sequences of biotin-labeled probes, hAR and mhAR. The probes were added to a lysate prepared from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins bound to the probes were captured by streptavidin magnetic beads and detected by immunoblot analysis using anti-Sp1, anti-TAF4 and anti-Rta antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 hr (lytic) or treated with DMSO (latent) were fixed with formaldehyde and the DNA-protein complexes were immunoprecipitated with anti-Sp1, anti-TAF4 and anti-Rta antibodies. The binding of Sp1, TAF4 and Rta to the hAR promoter was investigated by qPCR. (C) 293T cells were cotransfected with pCMV-R and TAF4 shRNA (empty column) or control shRNA (filled column) for 48 hr. CHIP assay was subsequently performed. The reaction with added anti-IgG antibody was used as a negative control. Error bar represents standard error. (D) 293T cells were cotransfected with pCMV-R and a reporter plasmid including phAR-luc or pmhAR-luc, in the presence of control shRNA (Ct-shRNA) (filled column) or TAF4 shRNA (empty column). Luciferase activities were monitored at 48 hr after transfection. Each transfection experiment was performed at lease three times, and each sample in the experiment was prepared in duplicate. The p value from each experiment was analyzed statistically with the Student's t- test method. Luc: luciferase gene.

Article Snippet: Monoclonal mouse anti-Rta antibody (1∶500 dilution) (Argene), or mouse anti-TAF4 antibody (1∶1000) (BD Biosciences) was added to the supernatant and incubated at 4°C for 1 hr.

Techniques: Plasmid Preparation, Labeling, Magnetic Beads, Western Blot, Affinity Precipitation, Immunoprecipitation, Binding Assay, shRNA, Negative Control, Luciferase, Transfection