mcaf1  (Novus Biologicals)

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Interaction among Sp1, p621 and Rta in vitro . GST–p621 was added to the lysate prepared from P3HR1 cells treated with TPA and sodium butyrate. Proteins bound to GST–p621 were pulled down with glutathione–Sepharose beads (Beads) and analyzed by IB using anti-Rta ( A ) and anti-Sp1 antibody ( B ). Parallel experiments involving GST–Rta (lane 3) and GST–Sp1 (lane 4) were also conducted and IB was performed with anti-MCAF1 antibody ( C ). Meanwhile, anti-HA antibody (lanes 2 and 3) and anti-Sp1 antibody (lanes 4 and 5) were added to mixtures containing Sp1, GST–p621 and Rta to determine whether the interaction between Sp1 and Rta requires p621 ( D ). Various amounts of Rta were added to binding mixtures that contained Sp1 and GST–p621. Proteins were pulled down using GST–p621–glutathione–Sepharose beads and analyzed by IB with anti-Sp1 antibody ( E ). In each part, lane 1 was loaded with cell lysate; in lane 2, proteins were eluted from GST–glutathione–Sepharose beads.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: In Vitro, Binding Assay

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Coimmunoprecipitation of Sp1, MCAF1 and Rta. Anti-Rta and anti-MCAF1 antibody were added to the lysates prepared from 293T cells transfected with pCMV-R and pcDNA-MCAF1 ( A and B ) or from P3HR1 cells that were treated with TPA and sodium butyrate ( C and D ). Proteins in the lysate immunoprecipitated by the anti-Rta antibody, anti-MCAF1 antibody or anti-Sp1 antibody were analyzed by IB with anti-Rta antibody (A–D, lanes 1–4), anti-MCAF1 antibody (A and C, lanes 5–8) and anti-Sp1 antibody (B and D, lanes 5–8). Reactions involving anti-IgG antibody were used as negative controls.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Transfection, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Indirect immunofluorescence analysis. P3HR1 cells were transfected with pCMV-R ( A – D , I – L ) or an empty vector ( E – H , M – P ). Cells were incubated with monoclonal anti-Rta antibody (B, F, J and N), rabbit anti-MCAF1 polyclonal antibody (C and G) and rabbit anti-Sp1 polyclonal antibody (K and O). DAPI staining revealed the positions of the nucleus. Finally, cells were examined using a confocal laser-scanning microscope. D, H, L and P are merged images.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Incubation, Staining, Laser-Scanning Microscopy

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Binding of the Sp1, MCAF1 and Rta to an Sp1-binding site on Rp. A biotin-labeled double-stranded Sp1 probe (Rp64/28) that contains the sequence from −64 and −28 of Rp was added to a lysate prepared from P3HR1 cells treated with TPA and sodium butyrate. A probe with an identical sequence, except for a mutated Sp1-binding sequence (Rpm64/28), was used as a negative control. Proteins bound to the probes were captured with streptavidin magnetic beads, extracted and detected by IB with anti-Sp1, anti-MCAF1 and anti-Rta antibodies.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Binding Assay, Labeling, Sequencing, Negative Control, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: CHIP analysis of the binding of Sp1, MCAF1 and Rta to an Sp1 site on Rp. P3HR1 cells were cotransfected with pCMV-R, pcDNA-MCAF1 and pR-Sp1 (lanes 1–5). Meanwhile, in separate experiments, pR-mSp1 (lanes 6–10) rather than pR-Sp1, was cotransfected. Binding of Sp1, MCAF1 and Rta to the Sp1 site on pR-Sp1 was studied by CHIP analysis using anti-IgG antibody (lanes 2 and 7), anti-Sp1 antibody (lanes 3 and 8), anti-MCAF1 antibody (lanes 4 and 9) and anti-Rta antibody (lanes 5 and 10). Meanwhile, in positive controls, DNA in the cell lysate was used as a template for PCR to amplify the Rp sequence (lanes 1 and 6); Rp, an Rp fragment amplified from pR-Sp1; M, 100 bp ladder.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Binding Assay, Sequencing, Amplification

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Mapping the interaction domains on MCAF1 and Rta. Plasmid that expresses GFP-MCAF1 and its deletion derivatives ( A ) was cotransfected with pCMV-R into 293T cells. ( B ) Proteins in the lysates prepared from cells that express GFP (lanes 1 and 5), GFP-MCAF-N (lanes 2 and 6), GFP-MCAF1-D1 (lanes 3 and 7) and GFP-MCAF1-D2 (lanes 4 and 8) were analyzed by IB using anti-GFP antibody (Input) and coimmunoprecipitation (Co-IP) with anti-Rta antibody and studied by IB with anti-GFP antibody. ( C ) Plasmids that express Flag-Rta, HA-Rta, GFP-Rta or Rta deletion mutants were cotransfected with pcDNA-MCAF1 into 293T cells. ( D ) Cell lysates that contain Flag-Rta (lane 2), Flag-Stu (lane 3), Flag-HincII (lane 4), Flag-HinfI (lane 5), Flag-EcoRII (lane 6), HA-Rta (lane 8), HA-RN290 (lane 9), GFP-Rta (lane 11) and GFP-R255/290 (lane 12) were immunoprecipitated with anti-MCAF1 antibody and analyzed by IB with anti-Rta antibody (lanes 1–6), anti-HA antibody (lanes 7–9) and anti-GFP antibody (lanes 10–12). Lanes 1, 7 and 10 were loaded with the proteins from the cells transfected with an empty vector.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation, Transfection

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Involvement of MCAF1 in Rp autoregulation. Reporter plasmids, pRp ( A ), pR-Spl ( B ) and pRRE ( C ) were cotransfected with pCMV-R and pcDNA-MCAF1 into P3HR1 cells. ( D ) Displays the luciferase activity expressed by pRRE and pR-Sp1 after cotransfecting pCMV-R. Luciferase activity was determined at 24 h after transfection. Each transfection experiment was performed at least three times, and each sample in the experiments was prepared in duplicate. The values obtained from the experiment were analyzed statistically by the least square means method. *, P < 0.05; **, P < 0.01.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Luciferase, Activity Assay, Transfection

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Activation of the expression of EBV immediate-early genes by MCAF1. ( A ) P3HR1 cells (5 × 10 6 ) were treated with TPA and sodium butyrate to activate the expression of Rta (lane 1). Cells untreated with TPA and sodium butyrate were transfected with 20 µg of pcDNA3.1 (lanes 2) and pcDNA-MCAF1 (lanes 3). Cotransfection was also performed by transfecting 0.5 µg of pCMV-R and 1–20 µg of pcDNA-MCAF1 (lanes 4–7). Lysates were prepared at 48 h after transfection. Immunoblot analysis was performed with anti-Rta and anti-α-tubulin antibodies. ( B ) 293 cells (5 × 10 6 ) that were infected by maxi-EBV (lane 2) were transfected with 20 µg of pcDNA3.1 (lanes 3 and 4) and pcDNA-MCAF1 (lanes 5 and 6). Proteins were prepared from the cells at 24 h (lanes 3 and 5) and 48 h (lanes 4 and 6) after transfection. Lane 1 was loaded with the lysate from 293 cells. Immunoblot analysis was performed using anti-Rta, anti-Zta and anti-α-tubulin antibodies.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Activation Assay, Expressing, Transfection, Cotransfection, Western Blot, Infection

Journal: Nucleic Acids Research

Article Title: Activation of Sp1-mediated transcription by Rta of Epstein–Barr virus via an interaction with MCAF1

doi: 10.1093/nar/gki956

Figure Lengend Snippet: Transcriptional activation of Rp, an Sp1 promoter and an RRE promoter by Rta. ( A ) 293T cells were transfected with pCMV-R and the lysate was mixed with biotin-labeled probes, RRE and mRRE. Proteins bound to the probe were captured using streptavidin magnetic beads. Proteins extracted from the beads were analyzed by IB with anti-Rta and anti-MCAF1 antibody. A similar study was performed with 293T cells transfected with plasmids that expressed HA-Rta (HA-R) and Rta(K213A). ( B ) An empty vector (HA) and plasmids that expressed HA-Rta (HA-R) and Rta(K213A) (K213A) were cotransfected with reporter plasmids pR-Spl, pSp1-luc, pRRE and pGL2-Basic (closed rectangle). Meanwhile, pcDNA-MCAF1 was also cotransfected to examine whether MCAF1 influenced the transactivation activity of pRRE (open rectangle). The nonspecific activation of the transcription of the luciferase gene in pGL2-Basic by Rta and Rta(K213A) was probably attributed to a GGGAGG sequence, which resembles an Sp1-binding site, near the cloning site of the plasmid. Luciferase activity was determined at 24 h after transfection. Each transfection experiment was performed at least three times, and each sample in the experiments was prepared in duplicate.

Article Snippet: Plasmid pEGFP-MCAF1-N, which contains the N-terminal 561 amino acids of MCAF1, was constructed by inserting a PCR-amplified fragment into the HindIII and SmaI sites of pEGFP-C1 (Clontech).

Techniques: Activation Assay, Transfection, Labeling, Magnetic Beads, Plasmid Preparation, Activity Assay, Luciferase, Sequencing, Binding Assay, Clone Assay

Journal: PLoS ONE

Article Title: MCAF1 and Rta-Activated BZLF1 Transcription in Epstein-Barr Virus

doi: 10.1371/journal.pone.0090698

Figure Lengend Snippet: P3HR1 cells were treated with TPA and sodium butyrate for 24(A) Proteins in the lysate were immunoprecipitated with anti-ATF2 antibody (lanes 3, 8), anti-Rta antibody (lanes 4, 7) or anti-IgG antibody (lanes 2, 6). Immunoblot analysis was performed using anti-Rta (lanes 1–4) and anti-ATF2 antibodies (lanes 5–8). (B) Proteins in the lysate from P3HR1 cells were immunoprecipitated with anti-IgG antibody (lanes 2, 6), anti-ATF2 antibody (lanes 3, 8), or anti-MCAF1 antibody (lanes 4, 7). Immunoprecipitated proteins were detected by immunoblotting, using anti-MCAF1 antibody (lanes 1–4) or anti-ATF2 antibody (lanes 5–8). Lanes 1 and 5 in (A) and (B) were loaded with 1% of total protein from the cell lysate. (C) GST-ATF2 (lane 5) or GST (lane 4) was added to the lysate prepared from E. coli BL21(DE3)(pET-MCAF1) (lane 1). Proteins bound to GST-ATF2 were pulled down by glutathione-Sepharose beads, and analyzed by immunoblotting (IB) with anti-His antibody (lanes 1–3). Lane 1 was loaded with 1% of cell lysate. GST or GST-ATF2 bound to glutathione-Sepharose beads were analyzed by immunoblotting (IB) with anti-GST antibody (lanes 4, 5).

Article Snippet: Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies.

Techniques: Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: MCAF1 and Rta-Activated BZLF1 Transcription in Epstein-Barr Virus

doi: 10.1371/journal.pone.0090698

Figure Lengend Snippet: (A) A biotin-labeled double-stranded ZII probe, containing the ATF2-binding sequences in Zp, was added to a lysate prepared from P3HR1 cells treated with TPA and sodium butyrate. A mutant probe, mZII, which contains a mutated ATF2-binding sequence, was used as a negative control. Proteins bound to the probes were captured using streptavidin magnetic beads, and were then extracted and detected by immunoblotting with anti-Rta, anti-MCAF1, and anti-ATF2 antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells treated with TPA and sodium butyrate for 48 h (lytic), or DMSO (latent), were analyzed by ChIP assay using anti-Rta, anti-MCAF1 and anti-ATF2 antibodies, with anti-IgG antibody used as a negative control. The binding capabilities of ATF2, MCAF1, and Rta to the ZII region in Zp were examined by qPCR. qPCR with primers specific for the BcLF1 intergenic region was used as a negative control. The error bar represents standard error. The p values from each experiment were derived using the Student's t- test. * p< 0.05.

Article Snippet: Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies.

Techniques: Labeling, Binding Assay, Mutagenesis, Sequencing, Negative Control, Magnetic Beads, Western Blot, Affinity Precipitation, Derivative Assay

Journal: PLoS ONE

Article Title: MCAF1 and Rta-Activated BZLF1 Transcription in Epstein-Barr Virus

doi: 10.1371/journal.pone.0090698

Figure Lengend Snippet: Plasmids expressing GFP-MCAF1-N (lanes 2, 7), GFP-MCAF1-DM1 (lanes 3, 8), GFP-MCAF1-M (lanes 4, 9), or GFP-MCAF1-DM2 (lanes 5, 10) were transfected into 293T cells. Plasmid pEGFP-C1 (lanes 1, 6) transfectants were used as a control (A). After transfection, cells were treated with TPA, and proteins in the lysates were immunoprecipitated (IP) using anti-ATF2 antibody. Detection by immunoblotting (IB), using anti-GFP or anti-ATF2 antibodies, was subsequently performed (B). Additionally, lysates prepared from E. coli expressing either His-ATF2 (lanes 2, 7), His-ATF2-N1 (lanes 3, 8), His-ATF2-N2 (lanes 4, 9), or His-ATF2-C (lanes 5, 10) (C) were mixed with the lysates prepared from 293T cells transfected with pcDNA-MCAF1 (lane 11). Proteins immunoprecipitated with anti-Flag antibody (lanes 12–16) were detected by immunoblotting with anti-His (lanes 6–10) or anti-Flag (lanes 12–16) antibodies. E. coli BL21 (pET32a) lysate was used as a negative control (lanes 1, 6) (D). Input lanes in 4B and 4D were loaded with 1% of the cell lysate.

Article Snippet: Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies.

Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: MCAF1 and Rta-Activated BZLF1 Transcription in Epstein-Barr Virus

doi: 10.1371/journal.pone.0090698

Figure Lengend Snippet: (A) His-tagged proteins, including Rta, ATF2, and MCAF1, were expressed in E. coli BL21(DE3). Lysates were mixed in different combinations for immunoprecipitation (IP) using anti-ATF2 antibody, and proteins bound to protein A/G agarose beads were then detected by immunoblotting (IB) using anti-Rta or anti-ATF2 antibodies. Lane 1 was loaded with 1% His-Rta. The reaction was also conducted using anti-IgG as a control. (B) 293T cells were cotransfected with pCMV-R and either MCAF1 siRNA or control siRNA. After transfection, cells were immunoprecipitated (IP) by anti-ATF2 (lanes 4, 6) or anti-IgG antibody (lanes 3, 5), and detected by immunoblotting (IB) using anti-Rta antibody. The effect of MCAF1 siRNA on the expression of MCAF1 was examined by immunoblotting using anti-MCAF1 and anti-ATF2 antibodies (lanes 1, 2). Lanes 1 and 2 were loaded with 1% of total protein from cell lysates.

Article Snippet: Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies.

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: PLoS ONE

Article Title: MCAF1 and Rta-Activated BZLF1 Transcription in Epstein-Barr Virus

doi: 10.1371/journal.pone.0090698

Figure Lengend Snippet: (A) 293T cells were cotransfected with pCMV-R and a reporter plasmid, pNS1-ZII, pNS1-ZIIM, or pNS3, as well as control siRNA or MCAF1 siRNA. Luciferase activity exhibited by the cells was examined at 48 h after transfection. The reduction of MCAF1 by siRNA was examined by immunoblotting with anti-MCAF1 antibody. Anti-α-tubulin antibody was used as a control. In addition, 293T cells were cotransfected with pCMV-R and a reporter plasmid, pNS1-ZII, pNS1-ZIIM, or pNS3, as well as varying amounts (µg) of pEGFP-ATF2-C (B) or pEGFP-ATF2 (69A/71A) (C). The expression of GFP-ATF2-C (B) or GFP-ATF2 (69A/71A) (C) was examined by immunoblotting, using anti-GFP antibody. Promoter activities were determined at 48 h after transfection. Each transfection experiment was performed at least three times, and all samples were prepared in duplicate. The p values from each experiment were derived using the Student's t-test.

Article Snippet: Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies.

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Transfection, Western Blot, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: MCAF1 and Rta-Activated BZLF1 Transcription in Epstein-Barr Virus

doi: 10.1371/journal.pone.0090698

Figure Lengend Snippet: (A) The BMRF2 reporter plasmid and a mutant version. 293T cells were cotransfected with pCMV-R and a reporter plasmid, pBMRF2-AP1, pBMRF2-mAP1, or pGL2-Basic. Luciferase activities were examined at 24 h after transfection. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 h (lytic) or DMSO (latent) were analyzed by ChIP assay using anti-Rta, anti-MCAF1 and anti-ATF2 antibodies. Anti-IgG antibody was used as a control. The binding of ATF2, MCAF1 and Rta to the AP-1 sequence in the BMRF2 promoter was examined by qPCR. Error bars represent standard error. The p values from each experiment were derived using the Student's t-test. * p< 0.05, ** p <0.001. Luc: luciferase gene.

Article Snippet: Proteins in the lysate were immunoprecipitated with anti-Rta (Argene), anti-MCAF1 (Invitrogen), anti-ATF2 (Abcam), or anti-Flag (Sigma) antibodies.

Techniques: Plasmid Preparation, Mutagenesis, Luciferase, Transfection, Binding Assay, Sequencing, Derivative Assay

Journal: Nature Communications

Article Title: In vivo epigenetic editing of Sema6a promoter reverses transcallosal dysconnectivity caused by C11orf46/Arl14ep risk gene

doi: 10.1038/s41467-019-12013-y

Figure Lengend Snippet: C11orf46 binds to the SETDB1 complex. a Flow diagrams for immunoaffinity purification of C11orf46 followed by mass spectrometric analysis. b Eight clones of inducible HEK293 cell lines expressing FLAG-tagged C11orf46 (clone 1–5 and 10) and a truncated form C11orf46 lacking amino acids 209–237 at the C-terminal region which corresponds to cysteine rich domain (CRD) of C11orf46 (C11orf46∆) (clone 7 and 8) in the presence of doxycycline (Dox+). SETDB1 was co-precipitated with C11orf46, but not with C11orf46∆. c Silver staining of proteins co-precipitated with C11orf46 (clone 10) or C11orf46∆ (clone 8). SETDB1, MCAF1 and KAP1 were detected in co-precipitate from C11orf46, but not of C11orf46∆. d Top scoring proteins co-precipitated with C11orf46 in cell lines overexpressed with C11orf46, C11orf46∆, and control cells are shown with % of amino acid sequence coverage. e Protein abundance in affinity purified C11orf46 complexes. Notice prominence of SETDB1-MCAF1-KAP1 repressor proteins (black). Bar graphs indicate mean ± S.E.M. n = 3 independent quantification. f Schematic diagrams indicating position of C11orf46 arginine to histidine substitution at codon 236 (R236H) in conserved cysteine rich domain (CRD; amino acids 209–237) between human and mouse. g , h Co-immunoprecipitation experiments using protein lysates from HEK293 cells overexpressing FLAG-tagged C11orf46, C11orf46∆, or C11orf46 R236H showed that absence of CRD domain and R236H mutation is associated with weakened or non-detectable C11orf46-SETDB1 binding and partial loss of binding to other components. R236H mutation does not fully disrupt C11orf46-KAP1 binding. i R236H mutation and CRD deletion of C11orf46 do not affect binding to histone H3. j Flow diagrams for immunoaffinity purification of SETDB1 followed by mass spectrometric analysis. k Inducible SETDB1 expression system in HEK293 cell lines (48 h after Dox treatment). l , m Silver staining of proteins co-precipitated with SETDB1. Both endogenous C11orf46 and SETDB1 binding partners were detected by mass spectrometry. Top scoring proteins co-precipitated with SETDB1 in cell lines overexpressed with SETDB1 and control cells are shown. n ( left) C11orf46-SETDB1 co-immunoprecipitation in human cerebral cortex. (right) SETB1 co-immunoprecipitates include C11orf46, HP1γ, and MCAF1 in HeLa nuclear extract. o Schematic summary of above data representing working model of C11orf46-SETDB1 complex in relation to histone H3

Article Snippet: For immunoblots, the following antibodies were used: rabbit monoclonal antibody against the C-terminus of human SETDB1 (Cell Signaling technology, Cat #2196S), KAP1 (cell signaling technology, Cat#4124S); rabbit polyclonal antibody against SETDB1 (Santa Cruz, Cat #sc-66884X), MCAF1 (Novus biological, Cat #NB100-438), Histone H3 C-terminus (Millipore, Cat #07-690).

Techniques: Immunoaffinity Purification, Clone Assay, Expressing, Silver Staining, Control, Sequencing, Quantitative Proteomics, Affinity Purification, Immunoprecipitation, Mutagenesis, Binding Assay, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: Binding of Zta, MCAF1 and Rta to ZRE. ( A ) Probe Rp-SZ contains the sequence from −64 to −28 in the BRLF1 promoter. It also includes an Sp1 site (oval) and ZRE (rectangle). Probes Rp-mS, Rp-mZ and Rp-mSZ have the same sequence as Rp-SZ, except that the Sp1 site and ZRE in Rp-SZ were mutated as indicated by a crossed rectangle and a crossed oval. The number represents the nucleotide position relative to the transcriptional start site, ‘+1’ (arrow). ‘TATA’ represents TATA box. ( C ) A probe, MRP-RZ, contained the region between −174 and −36 in the BMRF1 promoter. Probes MRP-mR, MRP-mZ and MRP-mRZ contained a mutated RRE, ZREs and both RRE and ZREs as indicated by crossed rectangles. ( E ) Probe HLp-Z contained a ZRE from the BHLF1 promoter. In HLp-Z, the ZRE was mutated. ( B, D, E ) The probes were added to a lysate from P3HR1 cells that had been treated with TPA and sodium butyrate. Proteins that were bound to the probes were captured using streptavidin magnetic beads and analyzed by immunoblotting with anti-MCAF1, anti-Rta, anti-Sp1 and anti-Zta antibodies.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

Techniques: Binding Assay, Sequencing, Magnetic Beads, Western Blot

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: Interaction among Zta, MCAF1 and Rta in vivo . ( A ) P3HR1 cells were treated with TPA and sodium butyrate. Proteins in the lysate (lanes 1 and 5) were immunoprecipitated (IP) with anti-IgG (lanes 2 and 6), anti-MCAF1 (lanes 4 and 7) and anti-Zta (lanes 3 and 8) antibodies. Immunoprecipitated proteins were analyzed by immunoblotting (IB) with anti-MCAF1 (lanes 1–4) and anti-Zta (lanes 5–8) antibodies. ( B ) Proteins in the lysate (lanes 1 and 5) were immunoprecipitated with anti-IgG (lanes 2 and 6), anti-Rta (lanes 3 and 7) and anti-Zta (lanes 4 and 8) antibodies. Immunoprecipitated proteins were detected by immunoblotting with anti-Rta (lanes 1–4) and anti-Zta (lanes 5–8) antibodies. Lane 1 in (A) and (B) was loaded with 1% of the cell lysate. Lane 5 in (A) and (B) was loaded with 3% of the cell lysate.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

Techniques: In Vivo, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: Quantitation of the binding of Zta, MCAF1, and Rta to the ZREs in the promoters of BHLF1 ( A ), BHRF1 ( B ), BMRF1 ( C ) and BMLF1 ( D ). P3HR1 cells were treated with TPA and sodium butyrate. The binding of Zta, MCAF1 and Rta to the ZREs was investigated by CHIP analysis using anti-Zta, anti-MCAF1 and anti-Rta antibodies and by qPCR. The reaction was performed with and without the addition of anti-IgG antibody as negative controls. The error bar represents standard error.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

Techniques: Quantitation Assay, Binding Assay

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: Subcellular localization of MCAF1, Rta and Zta. P3HR1 cells were transfected with pEGFP-Rta ( A–D ) or an empty vector, pEGFP ( E–H ), and treated with TPA and sodium butyrate (SB). Cells were also treated ( I–L ) or untreated ( M–P ) with TPA and sodium butyrate without transfection. Cells were incubated with monoclonal anti-Zta antibody (C, G, K and O) and rabbit anti-MCAF1 polyclonal antibody (J and N). DAPI staining revealed the nucleus (A, E, I, M). Cells were observed under a confocal laser-scanning microscope. D, H, L and P are merged images.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

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

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: Mapping the interaction domains in MCAF1 and Zta. ( A ) Various regions in Zta were fused with GFP. Numbers represent the positions of amino acid in Zta. TA, transactivation domain; DBD, DNA-binding domain; DIM, dimerization domain. ( B ) Plasmids that expressed GFP-ZN (lanes 1 and 4), GFP-ZM (lanes 2 and 5) and GFP-ZC (lanes 3 and 6) were transfected into 293T cells. Proteins in the lysates were analyzed by immunoblotting (IB) with anti-GFP antibody (lanes 1–3). Proteins in the lysate were immunoprecipitated with anti-MCAF1 antibody and examined by IB with anti-GFP antibody (lanes 4–6). ( C ) Plasmids that expressed a segment of MCAF1 that was fused with GFP were used to identify the region in MCAF1 that interacted with Zta. Numbers represent positions of amino acids in MCAF1. Domains 1 and 2 were delineated by Fujita et al. . ( D ) The plasmids were transfected into P3HR1 cells that were treated with TPA and sodium butyrate. Proteins in the cell lysate that contained GFP-MCAF1-N (lane 1), GFP-MCAF1-DM1 (lane 2), GFP-MCAF1-M (lane 3) and GFP-MCAF1-DM2 (lane 4) were detected by IB with anti-GFP antibody (lanes 1–4). Proteins in the lysate were immunoprecipitated with anti-Zta antibody and analyzed by immunoblotting with anti-GFP antibody (lanes 5–8). ( E ) The lysate from E. coli BL21(DE3)(pET-Zta) was mixed with GST-Rta-, GST-MCAF1-C1-, GST-MCAF1-C2- or GST-glutathione-Sepharose-beads. Proteins that were bound to beads were detected by immunoblotting using anti-Zta antibody (lanes 1–5). E. coli lysates that contained His-Rta, His-MCAF1-C1 and His were mixed with GST-Zta-glutathione-Sepharose beads. Proteins that were pulled down by the beads were examined by IB with anti-Rta antibody (lanes 6–8). Lanes 1 and 6 were loaded with 0.5% of the lysate. Lane 9 shows the immunoblot of His-MCAF1-C1 that was purified by Ni-NTA agarose beads.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

Techniques: Binding Assay, Transfection, Western Blot, Immunoprecipitation, Purification

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: MCAF1 and synCergistic activation of transcription by Rta and Zta. ( A ) 293T cells were cotransfected with a reporter plasmid, pBHLF1 or pEAD, and an empty vector, pCMV-3 (CMV), plasmids that expressed Rta (R), Zta (Z) and MCAF1 (M). ( B ) Cells were also cotransfected with pCMV-R, pCMV-Z and 0–0.5 μg of pcDNA-MCAF1. ( C ) A similar study was conducted in BJAB cells. The luciferase activity of the cells was determined 48 h following transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

Techniques: Activation Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection

Journal: Nucleic Acids Research

Article Title: MCAF1 and synergistic activation of the transcription of Epstein–Barr virus lytic genes by Rta and Zta

doi: 10.1093/nar/gkq243

Figure Lengend Snippet: Knockdown of MCAF1 and synergistic activation. ( A ) 293T cells were transfected with MCAF1 siRNA (lane 2) or control siRNA (lane 1). The expression of MCAF1 was determined by immunoblotting using anti-MCAF1 antibody at 48 h following transfection. The amount of α-tubulin in the cell was used as a control. ( B ) 293T cells were cotransfected with pBHLF1, pEAD, or pBMLF1 with pCMV-R, pCMV-Z and MCAF1 siRNA or control siRNA. The luciferase activity of the cells was determined 48 h following transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate.

Article Snippet: To suppress MCAF1 expression, 293T cells (1 × 10 4 ) were transfected with 0.3 μg pBHLF1, pEAD and 50 pmol siRNA MCAF1 or control siRNA (Invitrogen primer number 130189C06) with Lipofectamin 2000 (Invitrogen).

Techniques: Activation Assay, Transfection, Expressing, Western Blot, Luciferase, Activity Assay