Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) FLAG-mouse ORC2 or ORC1 were transfected into C33A cells with either BPV-1 E2 or E2R (aa 162–410). 24 hr later, E2 immunoprecipitations (II-1 antibody) were blotted with E2 B201 or M2 antibodies. (B) ORC2 interacts with the BPV-1 E2 transactivation domain (TAD, aa 1–216). FLAG-mouse ORC2 was transfected into C33A cells with BPV-1 E2, TAD or E2R. 24 hr later, groups without full length E2 were treated with 10 μM MG132 for 6 hr to maintain input protein levels of ORC2. B201 antibody immune complexes were blotted with B201 and M2 antibodies. The TAD domain of E2 is the same size as light chain IgG as indicated by * in the Fig 1B. (C) HA-ORC2 was transfected into 293TT cells along with FLAG-HPV-31 E2. HA antibody immunoprecipitates were blotted with HA-7 and M2 antibodies. (D) HA-ORC2 was transfected into 293TT cells along with FLAG-HPV-31 E1 and FLAG-HPV-31 E2. HA antibody immunoprecipitations were blotted with ORC2 and FLAG antibodies. ORC2 was not detected in the inputs but was detected in the pull down. HA-ORC2 protein is approximately 70 kDa and FLAG-31E1 protein is approximately 75 kDa. (E) Human ORC2 protein was incubated with 6-His-BPV E2:1–216 and MBP-6-His-HPV-16 E6. Nickel complexes were blotted with E2 (B201), E6, and ORC2 antibodies.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Transfection, Incubation

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) In-situ proximity ligation assay (PLA) between FLAG-HPV-31 E2 and HA-hORC2. Fluorescent spots indicate interaction based DNA amplification occurring in the nucleus (stained blue). The no transfection group was used as a negative control with these antibodies. (B) PLA between FLAG-HPV-31 E2 and HA-HPV-31 E1. (C) PLA between FLAG-HPV-31 E2 and endogenous ORC2. Secondary antibodies only were used as a negative control.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: In Situ, Proximity Ligation Assay, Amplification, Staining, Transfection, Negative Control

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) Baculovirus expressing human ORC2 protein was added to 0.5% NP-40 lysis buffer containing protein A and protein G agarose bead slurry, and either mouse anti-EE or an equal mix of mouse anti-ORC2 (MBL) and rat anti-ORC2 (Cell Signaling) antibodies. IPs were loaded on a SDS page gel and ORC2 was detected with the mouse anti-ORC2 antibody (MBL) by immunoblotting. (B) HPV-BP and (C) CIN612-9E cells were synchronized with 2.5 mM double thymidine. Inset diagram shows flow cytometry based cell cycle analysis. ChIP was performed using mouse anti-ORC2 antibodies (MBL). Real-time PCR was completed with primers to the HPV-16 or HPV-31 LCR, a known mammalian origin of replication (GM-CSF), and Exon 9, a non-specific DNA region used as a negative binding control. EE (non-specific IgG) did not bind to these regions of DNA in these experiments and is not shown here.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Expressing, Lysis, SDS Page, Western Blot, Flow Cytometry, Cell Cycle Assay, Real-time Polymerase Chain Reaction, Binding Assay

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) C33A were transfected with the FLAG-HPV-31 E2 expression vector. 24 hours later cells were lysed (0.5% NP-40). The lysate from cells untransfected and cells transfected with FLAG-HPV-31 E2 were added to lysis buffer containing protein A and protein G bead slurry and rabbit anti-HPV-31 E2 serum. IPs were immunoblotted with M2 antibodies. (B) CIN612-9E cells were synchronized with 2.5 mM double thymidine. ChIP experiments were performed using mouse anti-EE, an equal mix of rat and mouse anti-ORC2 (Cell Signaling/MBL) or rabbit anti-HPV-31 E2 antibodies. Real-time PCR was performed with primers listed in Materials and Methods located in and around the HPV-31 LCR. The locations of the primers are shown below the horizontal axis with the ori labeled. E2 binding sites are located between the LCR2 and LCR4 primer sets. Ct values are normalized to input and EE was set to equal 1. Values are expressed as mean +/- SEM. * p-value ≤ 0.05 compared to EE. (C) C33A cells were transfected with FLAG-HPV-31 E1 expression vector and lysates were immunoprecipiated with mouse anti-EE, rabbit anti-16 E1, and rat anti-16 E1 antibodies and then immunoblotted with M2 antibodies. (D) W12 cells were synchronized with 2.5 mM double thymidine. ChIP experiments were performed using mouse anti-EE, equal mix of rat and mouse anti-ORC2 antibodies (Cell Signaling/MBL), or rat anti-HPV-16 E1 antibodies. Real-time PCR was performed with primers listed in Materials and Methods located in and around the HPV-16 LCR. The locations of the primers are shown below the horizontal axis with the ori labeled. E2 binding sites are located between the LCR3 and E6 primer sets. Ct values are normalized to input and EE was set to equal 1. Values are expressed as mean +/- SEM. * p-value ≤ 0.05 compared to EE.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Transfection, Expressing, Plasmid Preparation, Lysis, Real-time Polymerase Chain Reaction, Labeling, Binding Assay

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) Replication luciferase assays were completed with pFLORI31 and ORC2 depletion. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. ORC2 shRNA decreased ORC2 protein expression in C33A cells. (B) ORC2 shRNA decreased EBNA-1 based replication using the pREP4 plasmid (EBNA-1 based replication) in C33A cells. (C) ORC2 shRNA decreased ORC2 protein expression in HPV-BP and SiHa cells. (D) ORC2 knockdown increased HPV replication in cells containing HPV-16 episomes (HPV-BP) but not in cells with integrated HPV-16 (SiHa). HPV-BP and SiHa cells were transfected with 5.5 μg of shRNA hairpins. 7d later, cells were harvested and HPV-16 copy number (HPV-16 LCR) as normalized to GM-CSF ori #2. This experiment was performed three times with a representative image presented here.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Luciferase, shRNA, Expressing, Plasmid Preparation, Transfection

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) ORC2 siRNA duplexes decreased ORC2 protein levels at a concentration of 15 or 5 nM after 48 h. (B) CIN612-9E cells were transfected with RLuc and pFLORI31. 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (C) CIN612-9E cells were transfected with 15 nM ORC2 and differentiated in 10% FBS DMEM + 2 mM CaCl 2 for 48h. Involucrin and β-actin levels were analyzed. (D) CIN612-9E cells were transfected with RLuc and pFLORI31. Five hours later, the transfection was removed and cells were placed in either E-medium or 10% FBS DMEM + 2 mM CaCl 2 . 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (E) CIN612-9E cells were transfected with 15 nM control or ORC2 siRNAs, RLuc and pFLORI31. Five hours later, the transfection was removed and cells were placed in 10% FBS DMEM + 2 mM CaCl 2 . 48h later cells were lysed and luciferase activity measured. Values are expressed as mean +/- SEM. * p-value ≤ 0.05. (F) CIN612-9E cells were transfected with 15 nM control or ORC2 siRNAs and placed in 10% FBS DMEM + 2 mM CaCl 2 for 72 h. DNA was isolated and HPV-31 DNA content was measured by RT-PCR and normalized to β-actin levels. The ORC2 siRNA group was normalized to the control siRNA group (control siRNA = 1). Values are expressed mean +/-. * p-value ≤ 0.05.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Concentration Assay, Transfection, Luciferase, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: CIN612-9E cells were transfected with 30 nM of control or ORC2 siRNAs for 48 h. ChIP experiments were performed using (A) rabbit anti-HPV-31 E2 antibodies or (B) rat E1 antibodies. Real-time PCR was performed with primers listed in Materials and Methods located in HPV-31 LCR previously found to be enriched for E2 binding. E2 binding sites are located between the LCR2 and LCR4 primer sets. Ct values are normalized to input and values are expressed as mean +/- SEM. * p-value ≤ 0.05 compared to control siRNA.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Binding Assay

Journal: PLoS Pathogens

Article Title: The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

doi: 10.1371/journal.ppat.1005934

Figure Lengend Snippet: (A) Lysates (1% NP-40) for i31E2 cells ± Dox and control/HPV-16E2 U2OS cell lines were blotted with FLAG-M2 (HPV-31 E2), TGV261 (HPV-16 E2), ORC2 (MBL) and β-actin antibodies. (B,C) TRE-x U2OS cells containing pcDNA4/TO-FLAG 31E2 (i31E2, ± 48h Dox treatment) and Control and 16E2 U2OS cells were treated overnight with 2.5 mM thymidine, released the next day for 6h, followed by 2.5 mM thymidine treatment overnight. ChIP was performed with an equal mix of mouse/rat-ORC2 antibodies (MBL/Cell Signaling) on the GM-CSF ori (B) or the lamin B2 (LB) ori (C). ChIP experiments were performed at least three times. The Ct values for ORC2 binding were first normalized to input for each cell line. ORC2 levels for the E2 expressing cell lines were normalized to the control cell lines (no E2). Values are expressed as mean +/- SEM. * p-value ≤ 0.05 compared to control cell line.

Article Snippet: The following antibodies were used: Mouse anti-FLAG (M2; Sigma), mouse anti-HA (HA-7; Sigma 3F10; Roche, 12CA5), mouse anti-human ORC2 (MBL), rat anti-human ORC2 (Cell Signaling), mouse anti-HPV-16 E6 (Arbor Vita), mouse anti-EE and anti-β-actin (Sigma).

Techniques: Binding Assay, Expressing

Journal: Science Advances

Article Title: FBXO42 facilitates Notch signaling activation and global chromatin relaxation by promoting K63-linked polyubiquitination of RBPJ

doi: 10.1126/sciadv.abq4831

Figure Lengend Snippet: ( A ) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding epigenetically modified proteins. Then, cell lysates were incubated with S-protein beads and blotted with antibodies against FLAG- or MYC-epitope tags. ( B ) WT and FBXO42 KO cells were cotransfected with cSFB-RBPJ– and Myc-tagged constructs encoding SWI/SNF complex proteins. Then, the cells were harvested and analyzed as described in (A). ( C ) Heatmap showing the differential interaction between chromatin factors and RBPJ in WT and FBXO42 KO cells as identified by MS. ( D ) Enrichment analysis of the differentially interacting proteins of heterochromatin components is shown on the basis of GO annotation. ( E ) Immunofluorescence detection of HP1α foci in WT and FBXO42 KO cells. Scale bars, 10 μm. ( F ) HP1α foci number and percentage of HP1α foci area were calculated using ImageJ software. ( G ) WT and FBXO42 KO cells were digested with micrococcal nuclease (MNase) for 3 min, and chromatin relaxation was monitored by the release of nucleosomes. ( H ) Chromatin association of the SWI/SNF subunits SMARCA2, SMARCA4, and SMARCC2 in WT and FBXO42 KO cells was analyzed using Western blotting after chromatin isolation. ORC2 served as the loading control. ( I ) WT and FBXO42 KO cells were digested with deoxyribonuclease (DNase) for 3 min and followed with agarose gel electrophoresis analysis. ( J ) Chromatin from WT and FBXO42 KO cells was isolated, and DNase I was digested and used as substrate for accessibility assay. ( K and L ) The heatmap view for ATAC-seq signal intensity at TSSs in WT and FBXO42 KO JURKAT cells. ( M ) ATAC-seq peaks; H3K4m1, H3K4m3, and H3K27ac ChIP-seq peaks; and DNase-seq peaks downloaded from ENCODE database at MYC locus were analyzed. (A), (B), and (E) to (J), n = 3. Quantitative data are presented as means ± SEM. P values were calculated using two-tailed Student’s t tests. * P < 0.05 and ** P < 0.01.

Article Snippet: The following primary antibodies were used: rabbit anti-RBPJ [5313S, Cell Signaling Technology (CST), RRID:AB_2665555], mouse anti-FBXO42 (TA800283, OriGene, RRID:AB_2625356), THE hemagglutinin (HA) Tag (A01244, GenScript), THE c-Myc Tag (A00704, GenScript), ANTI-FLAG M2 antibody (B3111, Sigma-Aldrich, RRID:AB_2910145), rabbit anti-ubiquitin (AF0306, Beyotime), rabbit anti–β-actin (AC026, ABclonal, RRID:AB_2768234), rabbit anti-LSD1 (YM0422, ImmunoWay), rabbit anti-SMARCA4 (ET1611-85, HUABIO), rabbit anti-SMARCA2 (ER65406, HUABIO), rabbit anti-SMARCC2 (ER62787, HUABIO), and rabbit anti-origin recognition complex subunit 2 (ORC2) (A15697, ABclonal).

Techniques: Construct, Modification, Incubation, Immunofluorescence, Software, Western Blot, Isolation, Agarose Gel Electrophoresis, ChIP-sequencing, Two Tailed Test