Journal: The Journal of Experimental Medicine

Article Title: Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

doi:

Figure Lengend Snippet: ( A ) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area ). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer ( BW ) and a set of locus-specific primers (1, 2 or 1′, 2′). ( B ) SBE in developing B cells from wild-type and Eμ- myc mice. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c and Eμ- myc mice was subjected to LMPCR to detect SBE upstream of the D FL16.1 , J κ1 , and J κ2 segments. PCR products were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The identities of the indicated PCR products were confirmed by DNA sequence analysis . Labeled products were visualized with a PhosphorImager. Lanes 1–10 , B cell development stages as defined in Table ; lane 11 , 6312, a pro-B cell line derived from a RAG-2-deficient mouse ; lane 12 , B220 + cells from bone marrow, representing all stages of B cell development; lane 13 , same as lane 12 , with no ligase added at the linker ligation step. The bottom strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus ( CD14 ), showing equivalent amounts of amplifiable DNA in all samples.

Article Snippet: The reaction was then mixed with 100 μl PCR-L buffer (10 mM Tris, pH 8.8, 50 mM KCl, 0.25% Tween-20, 0.25% NP-40) and heated to 95°C for 10 min. For PCR, the linker-ligated DNA was brought to 65°C, and 4 μl of each sample were added to 21 μl amplification mix containing the appropriate primers and Taq DNA polymerase (Life Technologies).

Techniques: Ligation, Amplification, Purification, Electrophoresis, Membrane, Hybridization, Sequencing, Labeling, Derivative Assay, Stripping Membranes, Staining, Agarose Gel Electrophoresis, Control

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Identification of the cohesin interactome with proximity ligation mass spectrometry. A Schematic of the experimental setup for cohesin proximity ligation followed by mass spectrometry. Transgenic NIH-3T3 mouse fibroblast cell lines were generated containing a 3xHA-TurboID or RAD21-3xHA-TurboID transgene with a tetracycline responsive element (TRE). Cell lines were treated with doxycycline for 36 h to promote expression of the transgenes. Additionally, RAD21-3xHA-TurboID cells that were not treated with doxycycline serve as a No TurboID control. Following doxycycline treatment, cells were treated ± biotin for 2 h. Cells were collected, lysed, and nuclear proteins were extracted. A streptavidin enrichment was performed, and a quality control check was done on 10% of the protein sample by western blotting for biotinylated proteins, while the remaining 90% of the streptavidin enriched sample was used for LC–MS/MS. Proteins identified by mass spectrometry were analyzed by two methods to identify proteins enriched relative to controls. Proteins enriched in the RAD21-3xHA-TurboID biotin treated sample relative to the: (1) 3xHA-TurboID biotin treated sample (Tier 1; p < 0.05, FC > 2); or (2) No TurboID biotin treated sample (Tier 2; p < 0.05, FC > 2). Each condition was performed in triplicate. B Co-purification of RAD21-3xHA-TurboID protein with endogenous cohesin subunits. IgG (negative control), anti-SMC3, and anti-HA antibodies were used to purify cohesin complexes in nuclear extracts from RAD21-3xHA-TurboID expressing cells under high stringency conditions. Cohesin subunits were detected by western blotting (SMC3, SMC1, RAD21, and HA). Arrows indicate endogenous RAD21 (black) and transgenic RAD21-3xHA-TurboID proteins (green). Box contains a depiction of core cohesin complex with RAD21-3xHA-TurboID. C Venn diagram showing the overlap of proteins detected in the Tier 1 cohesin interactome, Tier 2 cohesin interactome, and cohesin IP-MS experiments [ – ]. D , E Volcano plots showing proteins detected in the Tier 1 ( D ) and Tier 2 ( E ) cohesin interactomes. Proteins significantly enriched in Tier 1 and Tier 2 shown in red and blue, respectively. Known cohesin subunits and interactors are indicated with green dots and labels

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: Ligation, Mass Spectrometry, Transgenic Assay, Generated, Expressing, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Copurification, Negative Control, Protein-Protein interactions

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Cohesin co-localizes with SWI/SNF at enhancers, promoters, and CTCF sites. A Genome browser view of the Sox2 gene and super enhancer. ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) is shown. DNA interactions were detected by Micro-C and are shown at the top with a contact frequency heatmap and arcs for DNA loops (5 kb resolution). RAD21 peaks overlapping BRG1 peaks highlighted in purple, RAD21 peaks not overlapping BRG1 highlighted in green, and BRG1 peaks not overlapping RAD21 highlighted in orange. B Heatmaps showing ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) at sites where cohesin peaks do not overlap SWI/SNF peaks (top), sites where cohesin peaks overlap SWI/SNF peaks (middle), and sites where SWI/SNF peaks do not overlap cohesin peaks (bottom). Signal is z-score normalized. C UpSet plot of cohesin peaks overlapping CTCF, H3K27ac, H3K4me3, and/or BRG1

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: ChIP-sequencing

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Altered cohesin occupancy at CTCF sites and enhancers upon SWI/SNF perturbation . A The SWI/SNF cBAF complex and three small molecules that inhibit or lead to degradation of the BRG1 subunit (dark orange). The ACBI1 PROTAC (red) targets the bromodomain of BRG1, recruiting an E3 ubiquitin ligase for poly-ubiquitination and proteasomal degradation. PFI-3 (purple) targets the bromodomain of the BRG1 subunit. BRM014 (blue) targets the ATPase active site of BRG1. The levels of SWI/SNF subunits shown in light orange were assessed following ACBI1 PROTAC treatment. B Genome browser views of RAD21 peaks that increase (up) or decrease (down) following ACBI1 PROTAC treatment. ChIP-seq signal for RAD21 with DMSO or ACBI1 treatment, BRG1, H3K4me3, H3K27ac, and CTCF is shown. Differential RAD21 peaks are indicated with a gray box. C Violin plots showing differential RAD21 signal in ACBI1 PROTAC treated mESCs relative to DMSO treated mESCs. The RAD21 peaks with significantly increased signal following ACBI1 treatment are shown in red, while peaks with significantly decreased signal are shown in gray. D Violin plots showing BRG1 (orange), H3K27ac (pink), and CTCF (dark blue) ChIP-seq signal at all RAD21 peaks, peaks with increased RAD21 signal (up) following ACBI1 treatment, and peaks with decreased RAD21 signal (down) following ACBI1 treatment. Significance determined by non-parametric Mann–Whitney test (****p-value < 0.0001). E Percentage of differential RAD21 peaks (up on left, down on right) upon ACBI1 treatment that overlap with the anchors of DNA loops detected by Micro-C, and percentage of non-differential RAD21 peaks with similar RAD21 signal intensity shown as a control. F Same as ( C ) for PFI-3 treatment. The RAD21 peaks with significantly increased signal following PFI-3 treatment are shown in purple, while peaks with significantly decreased signal are shown in gray. G Same as ( D ) for all RAD21 peaks and peaks with significantly different RAD21 signal following PFI-3 treatment ( n.s. not significant, *p-value < 0.01). H Same as ( E ) for PFI-3 treatment. I Same as ( B ) for BRM014 treatment. DNA loop anchor and long-range DNA interaction is depicted on top. J Same as ( C ) for BRM014 treatment. The RAD21 peaks with significantly increased signal following BRM014 treatment are shown in blue. K Same as ( D ) for all RAD21 peaks and peaks with increased RAD21 signal (up) following BRM014 treatment (****p-value < 0.0001). L Same as ( E ) for BRM014 treatment

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: Ubiquitin Proteomics, ChIP-sequencing, MANN-WHITNEY, Control

Journal: Cell

Article Title: Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription

doi: 10.1016/j.cell.2019.06.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-RBM17 , Bethyl , A302-498A; RRID: AB_1966059.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Staining, Blocking Assay, Ligation, Purification, Gel Extraction, Imaging, Sequencing, Software

Journal: Cell

Article Title: Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription

doi: 10.1016/j.cell.2019.06.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-RBM15 , Bethyl , A300-821A; RRID: AB_2253435.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Staining, Blocking Assay, Ligation, Purification, Gel Extraction, Imaging, Sequencing, Software

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: The HECT-Type Ubiquitin Ligase TRIP12 Controls PARP1 Abundance (A) Schematic representation of WWE domain-containing ubiquitin E3 ligases. HECT, homologous to the E6-AP carboxyl terminus; RING, really interesting new gene. (B) Multiple sequence alignment of WWE domains present in ubiquitin E3 ligases. While RNF146, HUWE1, and TRIP12 each contain a single WWE domain, DELTEX1, DELTEX2, and DELTEX4 each contain a tandem WWE domain. The tryptophan and glutamate residues typical of the WWE domain are marked in red. (C) HeLa Kyoto PARP1-LAP cells expressing GFP-PARP1 from its natural promoter were transfected with negative control siRNA or with siRNAs against WWE-containing ubiquitin E3 ligases. Nuclear GFP-PARP1 levels were analyzed 72 h after siRNA transfection by high-content microscopy. Pooled results from two independent siRNAs against each target protein are shown. Mean (solid line) and standard deviation (SD) from the mean (dashed lines) are indicated. Representative single cell images are shown below. Scale bar, 10 μm. (D) Western blot analysis of GFP-PARP1 levels in HeLa Kyoto PARP1-LAP cells transfected with negative control siRNA or siRNA against TRIP12. GFP-PARP1 and poly(ADP-ribose) band intensities were quantified in Fiji and the fold change is indicated below. (E) Naive U-2 OS cells (first lane) and U-2 OS PARP1 knockout (KO) cells transfected with Myc-PARP1 and increasing amounts of GFP-TRIP12 (0, 1, 2, and 4 μg of plasmid, respectively) were analyzed for PARP1 levels by western blot. (F) U-2 OS transfected with GFP-TRIP12 were analyzed by high-content microscopy for GFP-TRIP12 and endogenous PARP1 levels. Nuclear GFP-TRIP12 and PARP1 intensities per cell are plotted, color-coded by GFP-TRIP12 expression. Representative single cell images are shown to the right. Scale bar, 10 μm. See also Figure S1 .

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Sequencing, Expressing, Transfection, Negative Control, Microscopy, Standard Deviation, Western Blot, Knock-Out, Plasmid Preparation

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: TRIP12 Limits PARylation and Affects Outcomes of PAR Signaling (A) U-2 OS cells were transfected with the indicated siRNAs and localized DNA damage was induced by laser micro-irradiation. Cells were allowed to recover for the indicated time periods and stained for γH2AX as DNA damage marker and PARP1. Representative single cell images are shown and quantification of PARP1 recruitment to sites of DNA damage is depicted below as mean ± SD. ∗∗∗, p < 0.001; ∗∗, p < 0.01. (B) U-2 OS cells were treated as in (A) and stained for γH2AX and PAR. Representative single cell images are shown and quantification of PAR formation at sites of DNA damage is depicted below as mean ± SD. ∗∗∗, p < 0.001; ∗∗, p < 0.01. (C) U-2 OS cells were treated as in (A) and stained for γH2AX and XRCC1. Representative single cell images are shown and quantification of XRCC1 recruitment to sites of DNA damage is depicted below as mean ± SD. ∗∗, p < 0.01. (D) U-2 OS cells were treated as in (A) and stained for γH2AX and MDC1. Representative single cell images are shown and quantification of MDC1 recruitment to sites of DNA damage is depicted below as mean ± SD. See also Figure S2 .

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Transfection, Irradiation, Staining, Marker

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: TRIP12 Interacts with and Poly-Ubiquitylates PARP1 in a PAR- and WWE-Dependent Manner (A) HEK293T cells were transfected with the indicated plasmids for co-immunoprecipitation (coIP) experiments, with or without prior PARPi treatment (olaparib: 10 μM, 1 h). FLAG-PARP1 was immunoprecipitated and the interaction with GFP-PARP1 was analyzed by western blot. IP samples were adjusted based on input levels of PARP1 to correct for the effect of TRIP12 on PARP1 abundance. (B) In vitro interaction assay of purified GST, GST-TRIP12-WWE wild type (WT), and GST-TRIP12-WWE R869A mutant with PARP1 and auto-PARylated PARP1. Recombinant purified PARP1 was incubated or not with a low (20 μM, +) or high (200 μM, ++) concentration of NAD + for 15 min at 30°C to induce PARP1 auto-PARylation prior to the GST interaction assay. PARP1 and PAR binding to the purified GST-fusion proteins was assessed by western blot. (C) Ex vivo interaction assay of purified GST, GST-TRIP12-WWE WT, and GST-TRIP12-WWE R869A mutant with whole cell lysates from HeLa cells, which were exposed to 1-mM H 2 O 2 for 15 min prior to cell lysis to induce PAR formation. PARG was depleted from these cells by siRNA to avoid the PARG-mediated rapid degradation of PAR. PARP1 and PAR binding to the purified GST-fusion proteins was assessed by western blot. (D) In vitro ubiquitylation assay with purified E1 (UBE1), E2 (UBE2L3), E3 (FLAG-TRIP12 WT, WWE mutant (R869A), or HECT mutant (C2034A) purified from HEK293T cells and ubiquitin, using auto-PARylated PARP1 as a target protein. PARP1 ubiquitylation was assessed by western blot with anti-ubiquitin antibody. TRIP12 levels were assessed by running the supernatant from the in vitro reaction via western blot. (E) In vivo ubiquitylation assay in HEK293T cells expressing FLAG-PARP1 and GFP-TRIP12 WT, WWE mutant (R869A), or HECT mutant (C2034A) together with HA-ubiquitin. FLAG-PARP1 was immunoprecipitated and PARP1 ubiquitylation was assessed by western blot. IP samples were adjusted based on input levels of PARP1 to correct for the effect of TRIP12 on PARP1 abundance. (F) In vitro ubiquitylation assay to monitor TRIP12 activity in absence or presence of purified PAR chains. FLAG-TRIP12 WT or the PAR-binding-deficient WWE mutant (R869A) were purified from HEK293T cells and incubated with E1 (UBE1), E2 (UBE2L3), and ubiquitin with or without different amounts of purified PAR chains as indicated. Auto-ubiquitylation of TRIP12 was assessed by western blot. See also Figure S3 .

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Purification, Mutagenesis, Recombinant, Incubation, Concentration Assay, Binding Assay, Ex Vivo, Lysis, Ubiquitin Assay, In Vivo, Expressing, Activity Assay

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: TRIP12 Controls PARP1 Stability and Proteasomal Degradation in a PAR-Dependent Manner (A) U-2 OS PARP1 KO cells were transfected with the indicated plasmids and the effect of GFP-TRIP12 WT or mutant expression on Myc-PARP1 levels was assessed by western blot. (B) U-2 OS cell were transfected with control siRNA or siRNA against TRIP12 for 48 h, followed by the indicated plasmid transfections for 24 h. Myc-PARP1 was immunoprecipitated and its ubiquitylation was assessed by western blot with an anti-HA antibody. Immunoprecipitation with a FLAG antibody was performed to detect unspecific binding. IP samples were adjusted based on input levels of PARP1 to correct for the effect of TRIP12 on PARP1 abundance. (C) U-2 OS were transfected with control siRNA or siRNA against TRIP12 and exposed to cycloheximide (CHX, 50 μM) for the indicated time periods. TRIP12 and PARP1 levels were assessed by western blot. PARP1 band intensities were quantified in Fiji and the fold changes are indicated below. (D) U-2 OS were transfected with control siRNA or siRNA against TRIP12 and exposed to a PARPi with low PARP1 trapping activity (PJ-34, 10 μM) as indicated for 16 h to block PARylation with minimal cytotoxicity. TRIP12 and PARP1 levels were assessed by western blot. (E) U-2 OS cells were transfected with control siRNA or siRNA against TRIP12 for 48 h, followed by the indicated plasmid transfections for 24 h. All TRIP12 plasmids were rendered siRNA-resistant by introducing multiple silent mutations. High-content imaging-derived violin plot show nuclear PARP1 intensities. Median (solid lines) and quartiles (dashed lines) are indicated. (F) U-2 OS PARP1 KO cells were transfected with plasmids for Myc-PARP1 and GFP-TRIP12 expression and exposed to the proteasome inhibitor MG132 (10 μM) as indicated. PARP1 levels were assessed by western blot. See also Figure S4 .

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Transfection, Mutagenesis, Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Binding Assay, Activity Assay, Blocking Assay, Imaging, Derivative Assay

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: TRIP12 Constrains PARP1 Trapping and Extenuates PARPi-Induced DNA Damage (A) U-2 OS cells transfected with control siRNA or siRNA against TRIP12 and exposed to MMS (0.01%) and PARPi (olaparib, 10 μM) for 1 h as indicated. Cells were pre-extracted and chromatin-bound PARP1 was analyzed by high-content microscopy. Violin plots with median (solid lines) and quartiles (dashed lines) are shown; unpaired t test was used for statistical analysis. Representative single cell images of endogenous chromatin-bound PARP1 are shown below. Scale bar, 10 μm. ∗∗∗∗, p < 0.0001. (B) U-2 OS cells transfected with control siRNA or siRNA against TRIP12 were treated with MMS (0.01%) and PARPi (olaparib, 10 μM) for 2 h as indicated. Cells were then subjected to chromatin fractionation to separate the soluble from the chromatin-bound fraction. Chromatin-bound PARP1 was analyzed by western blot. PARP1 band intensities were quantified in Fiji and the fold changes are indicated below. (C) HeLa Kyoto PARP1-LAP cells were transfected with control siRNA or siRNA against TRIP12 and exposed to PARPi (olaparib, 10 μM) for 16 h. QIBC-derived cell cycle resolved γH2AX profiles are shown. Representative single-cell images of γH2AX staining are shown below. Scale bar, 10 μm. (D) QIBC-derived EdU profiles are shown and percentages of cells in G1, S, and G2 are provided for the same cells analyzed in (C). (E) U-2 OS cells were transfected with siRNAs and exposed to PARPi (olaparib, 10 μM) as indicated. QIBC-derived cell cycle resolved profiles of nuclear 53BP1 foci are shown. Representative single-cell images of 53BP1 foci are shown to the right. Scale bar, 10 μm. (F) U-2 OS cells were treated as in (E) and QIBC-derived cell-cycle resolved profiles of nuclear RAD51 foci are shown. Representative single cell images of RAD51 foci are shown to the right. Scale bar, 10 μm. (G) U-2 OS cells were treated as in (E) and QIBC-derived cell-cycle resolved profiles of nuclear RPA foci are shown. Representative images of RPA foci are shown to the right. Scale bar, 10 μm. See also Figure S5 .

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Transfection, Microscopy, Fractionation, Western Blot, Derivative Assay, Staining

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: Downregulation of TRIP12 Sensitizes Cancer Cells to PARPi and Is Associated with Elevated PARP1 Abundance in Cancer Patients (A) U-2 OS cells were transfected with siRNAs and exposed to PARPi olaparib as indicated for clonogenic survival assays. Relative clonogenic survival in response to increasing doses of PARPi olaparib is shown. Means ± SD are depicted. (B) HeLa Kyoto PARP1-LAP cells were treated as in (A) and relative clonogenic survival in response to increasing doses of PARPi olaparib is shown. Means ± SD are depicted. (C) HCC1143 cells were treated as in (A) and relative clonogenic survival in response to increasing doses of PARPi olaparib is shown. Means ± SD are depicted. (D) MCF-7 cells were treated as in (A) and relative clonogenic survival in response to increasing doses of PARPi olaparib is shown. Means ± SD are depicted. (E) Scatterplot of adjusted PARP1 protein levels against the relative TRIP12 RNA expression in breast cancer, with BRCA mutant samples highlighted in red. Upper right shows p values and r in Pearson’s product-moment correlation tests. (F) As in (E) for the ovarian cancer samples. Upper right shows p values and r in Pearson’s product-moment correlation tests. See also Figure S6 .

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Transfection, RNA Expression, Mutagenesis

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet: Model for PAR-Targeted Ubiquitylation of PARP1 by TRIP12 and the Ensuing Proteasomal Degradation of PARP1 (A) TRIP12 binds auto-modified PARP1 via its WWE domain and triggers PARP1 polyubiquitylation and proteasomal degradation. (B) When TRIP12 expression or function is impaired, PARP1 accumulates to supra-physiological levels, resulting in enhanced PARP1 trapping and elevated PARPi susceptibility. See main text for further discussion.

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Modification, Expressing

Journal: Cell Reports

Article Title: The Ubiquitin Ligase TRIP12 Limits PARP1 Trapping and Constrains PARP Inhibitor Efficiency

doi: 10.1016/j.celrep.2020.107985

Figure Lengend Snippet:

Article Snippet: 800 μg of lysates were then incubated with 1 μg of rabbit anti-TRIP12 antibody (A301-814A, Bethyl) for 3 hours at 4°C.

Techniques: Recombinant, Modification, Protease Inhibitor, Ligation, Labeling, Purification, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

doi: 10.1101/2022.05.24.493324

Figure Lengend Snippet: a , Family pedigree with genotype of Chr8:107758095 G>C mutation in the OXR1 gene. b , Brain MRI. Mid-sagittal T1 weighted images showing marked cerebellar atrophy in patient II-4 at 6 years (left) and patient II-6 at 3 years and 4 months (middle), compared with the same view of a healthy control at 6-years (right). c , Genomic structure of human OXR1 and the mutation site. The OXR1 gene is located on chromosome 8 and consists of 19 exons, reference to ENSG000001164830 (Hg19). Triangle in magenta, the mutation in the donor splicing site of exon 18 within TLDc domain ( OXR1 ΔEx18 ), affecting all identified human OXR1 isoforms. d , cDNA sequence around the OXR1 mutation site of lymphoblasts from patient II-4 and healthy control. e , Western blot analysis of OXR1 level in lymphoblasts from patient II-4 and healthy control. β-actin was used as loading control.

Article Snippet: The lysate was pre-cleared by Dynabeads™ Protein A (Thermofisher Scientific, Cat#10001D) beads for 30 min. Add 1μg of purified rabbit anti IgG antibody (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) or 3μg OXR1 antibody (Bethyl Cat# A302-036A, RRID:AB_1576565) to the 1mg pre-clear cell lysate separately.

Techniques: Mutagenesis, Sequencing, Western Blot

Journal: bioRxiv

Article Title: Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

doi: 10.1101/2022.05.24.493324

Figure Lengend Snippet: a , Proliferation capacity of patient lymphoblasts ( OXR1 ΔEx18 ) as compared with control cells (CTRL). n=3 independent experiments, n=6 samples for each genotype in each experiment. b , Patient ( OXR1 ΔEx18 ) and control (CTRL) lymphoblasts were treated with indicated concentrations of hydrogen peroxide (H 2 O 2 ) for 1 h and harvested after 24 h recovery, populations of surviving (left), apoptotic (middle) and necrotic (right) cells determined by flow cytometry with FITC-Annexin V/PI double staining. n=3 independent samples. c , 8-oxoG levels in genomic DNA detected by mass spectrometry after H 2 O 2 treatment in patient ( OXR1 ΔEx18 ) and control (CTRL) lymphoblasts. n=3 independent samples in the first assay (0.25 mM), and n=6 independent samples in the second assay. d , Quantification of p21 and HO-1 protein expression measured by western blot analysis under regular culturing conditions of patient ( OXR1 ΔEx18 ) and control (CTRL) lymphoblasts. n=3 independent samples. e , Quantitative real-time PCR analysis of oxidative stress response genes in patient ( OXR1 ΔEx18 ) and control (CTRL) lymphoblasts exposed to H2O2 and recovery for 6 hours (R6h). n=3 independent samples. f , qPCR analysis of Caspase-9 ( CAS9 ) expression in patient ( OXR1 ΔEx18 ) and control (CTRL) lymphoblasts following 0.25 mM H 2 O 2 treatment and recovery for 3 hours (R3h) or 6 hours (R6h). n=3 independent samples. g , Western blot analysis and quantification of CAS9 expression and cleaved CAS9 (cCAS9) in patient ( OXR1 ΔEx18 ) and control (CTRL) lymphoblasts exposed to 0.25 mM H 2 O 2 and recovery for 3 hours (R3h) or non-treated (NT). β-ACTIN used as loading control, n=3 independent samples. h , Proliferation capacity of patient lymphoblasts with stable expression of OXR1D (LB -OXR1 ΔEx18 +pE-OXR1D) compared to patient lymphoblasts carrying only vector (LB- OXR1 ΔEx18 +vector), n=6 independent samples. i , Analyses of cell viability, apoptosis and necrosis patient lymphoblast samples stably expressing OXR1D (LB -OXR1 ΔEx18 +pE-OXR1D) or empty vector (LB- OXR1 ΔEx18 +vector) upon H 2 O 2 treatment.n=3 independent samples. All Data are shown as mean±SD. * p < 0.05, ** p < 0.01, Student’s t test.

Article Snippet: The lysate was pre-cleared by Dynabeads™ Protein A (Thermofisher Scientific, Cat#10001D) beads for 30 min. Add 1μg of purified rabbit anti IgG antibody (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) or 3μg OXR1 antibody (Bethyl Cat# A302-036A, RRID:AB_1576565) to the 1mg pre-clear cell lysate separately.

Techniques: Flow Cytometry, Double Staining, Mass Spectrometry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Plasmid Preparation, Stable Transfection

Journal: bioRxiv

Article Title: Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

doi: 10.1101/2022.05.24.493324

Figure Lengend Snippet: a , Immunocytochemical analyses of neurospheres from patient ( OXR1 ΔEx18 ) and healthy control (CTRL). Neurite outgrowth is visualized by antibody against neuron-specific class III beta-tubulin (Tuj1). Bar diagrams show quantification of the neurite (Tuj1 + ) covered area, the fluorescence intensity of covered area, and the longest neurite length by average of 5 longest neurites of each neurosphere (n = 15 independent neurospheres for each genotype). b , Principal components analysis (PCA) showing the clustering of samples based on principal component 1 and 2 (PC1 and PC2). All samples are three different clones of each genotype derived from the monolayer neuronal differentiation experiment. Induced pluripotent stem cells (iPSC) were collected at day0, neural aggregates (NA) at day2 (48h after initiation of differentiation) and neurons (Neu) at day20. c , Pathway enrichment analysis showing the core pathways involved in neural development by Gene Set Enrichment Analysis (GSEA, normalized enrichment score, |NES| ≥ 1; nominal p value, ( p ) < 0.05; false discovery rate (FDR) ≤ 0.25). d , Gene Ontology (GO) analysis by GSEA. The most underrepresented GO terms of biological processes in OXR1 ΔEx18 neurons are relevant to neural development. e , Heatmap showing z-score normalized read counts of the top ranked leading-edge genes in neurogenesis and differentiation that were differentially expressed in Neu_ OXR1 ΔEx18 . f , Heatmap showing z-score normalized read counts of the top ranked leading-edge genes in axon genesis and cell migration that were differentially expressed in Neu_ OXR1 ΔEx18 .

Article Snippet: The lysate was pre-cleared by Dynabeads™ Protein A (Thermofisher Scientific, Cat#10001D) beads for 30 min. Add 1μg of purified rabbit anti IgG antibody (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) or 3μg OXR1 antibody (Bethyl Cat# A302-036A, RRID:AB_1576565) to the 1mg pre-clear cell lysate separately.

Techniques: Fluorescence, Clone Assay, Derivative Assay, Migration

Journal: bioRxiv

Article Title: Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

doi: 10.1101/2022.05.24.493324

Figure Lengend Snippet: a , Proximity ligation assay showing the interaction of OXR1 with PRMT1 (magenta spots) and PRMT5 (green spots) in patient fibroblasts ( OXR1 ΔEx18 ) and control cells (CTRL). Nuclei are indicated by DAPI staining. b , The Western blot analysis showing histone arginine modifications in induced pluripotent stem cells (iPSC) and neural stem cells (NSC). The arginine modification marks include H3R2me2s, H3R2me2a, H3R17me2a, H4R3me2a, H4R3me2s, H3R17me2s, H3R26me2s, and lysine marks H3K4me3, H3K27me3, and H3K9me3; Histone 3 was used as loading control. c , In vitro methyltransferase assay showing a direct role of OXR1 in regulating the enzymatic activity of PRMT5. N-terminal H4 peptides (1-21aa) and the mutant ones H4-R3K (1-21aa) were used as substrates. Different molar ratio of PRMT5/MEP50 protein complex (calculated as tetramer) to the full length OXR1A or truncated ones, such as 4:1 (+), 1:1(++), and 1:4(+++), was applied in each reaction. The enzymatic activity of PRMT5 was shown by the CPM (Count per minute) value of 3 H-Methyl incorporation. All Data are shown as mean±SD, * p < 0.05, *** p < 0.001, Student’s t test.

Article Snippet: The lysate was pre-cleared by Dynabeads™ Protein A (Thermofisher Scientific, Cat#10001D) beads for 30 min. Add 1μg of purified rabbit anti IgG antibody (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) or 3μg OXR1 antibody (Bethyl Cat# A302-036A, RRID:AB_1576565) to the 1mg pre-clear cell lysate separately.

Techniques: Proximity Ligation Assay, Staining, Western Blot, Modification, In Vitro, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

doi: 10.1101/2022.05.24.493324

Figure Lengend Snippet: a , The progression of cerebral organoid development is illustrated (upper). Representative images (bright field, 40x) show the progression of cerebral organoids derived from each healthy control (CTRL) and patient ( OXR1 ΔEx18 ) iPSC clones from day 2 to day 15 (lower left). Black arrows point the expanding neuroepithelial formation in CTRL clones from day 8 and the poorly developed neuroepithelia buds observed in the OXR1 ΔEx18 cerebral organoids at day 15. The body size of cerebral organoids (lower right) is quantified by summing the products of bi-dimensional measurements (SPD). SPD = Longest diameter (LD) Δ longest diameter perpendicular to the LD. n=15-20 organoids of each clone, * p < 0.05. b , Immunohistochemical analyses of cerebral organoids at day 75 using the antibody specific for MAP2 (the marker of mature neurons, yellow) or TBR1(the marker of deep layer neurons, magenta). c , Immunohistochemical analyses of cerebral organoids at day 75 using antibodies specific for REELIN (yellow) and OXR1 (magenta), respectively. d , A schematic illustration showing the guided progression of region-specific brain organoids. e , Representative images (bright field, 40x) show the progression of hypothalamus derived from healthy control (CTRL) and patient ( OXR1 Δ Ex18 ) iPSCs. The starting cell number of each clone is indicated (100% refer to 10,000 iPSC cells per microwell of Aggrewell-800 plate on day 0). After hypothalamus patterning, the spheroids of OXR1 ΔEx18 clone 1 (c1) and OXR1 ΔEx18 clone 2 (c2) dissipated by day 5. f , Representative images (bright field, 40x) show the progression of hippocampus organoids. After hippocampus patterning, the spheroids of OXR1 ΔEx18 c2 dissipated by day 5. g , Representative confocal images showing the progression of SOX2 + neural progenitor cells (magenta) during the development of hippocampus organoids. h , Representative confocal images showing the progression of NeuN + mature neurons (grey) and DCX + immature neurons (green) during the development of hippocampus organoids. In all confocal images, the scale bar is 60 µm and the nuclei were visualized using DAPI (blue).

Article Snippet: The lysate was pre-cleared by Dynabeads™ Protein A (Thermofisher Scientific, Cat#10001D) beads for 30 min. Add 1μg of purified rabbit anti IgG antibody (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) or 3μg OXR1 antibody (Bethyl Cat# A302-036A, RRID:AB_1576565) to the 1mg pre-clear cell lysate separately.

Techniques: Derivative Assay, Clone Assay, Immunohistochemical staining, Marker

Journal: bioRxiv

Article Title: Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

doi: 10.1101/2022.05.24.493324

Figure Lengend Snippet: a-b , Representative bright-field images (40x) show the progression of cortical and midbrain organoids, derived from healthy control (CTRL) or and patient ( OXR1 Δ Ex18 ) iPSCs. The starting cell number of each clone is indicated (100% refer to 10,000 iPSC cells per microwell of Aggrewell-800 plate on day 0). After cortical patterning, the spheroids of OXR1 ΔEx18 clone 2 (c2) usually dissipated by day 5. After midbrain patterning, OXR1 ΔEx18 c2 showed less midbrain organoids generated at day 5. c , Immunohistochemical analyses showing the CTIP2 + deep layer neurons (magenta) formed above SOX2-enriched progenitor zones (yellow) of cortical organoids at day 50. d , Immunohistochemical analyses showing the Ki67 + proliferating cells (green) and cCas3 + apoptotic cells (magenta) at VZ/SVZ progenitor zones of cortical organoids at day 50. The percentage of Ki67 + or cCas3 + cells in VZ/SVZ is quantified as shown in the bar diagram. n = 6 organoids. VZ, ventricular zone; SVZ, subventricular zone. e , Immunohistochemical analyses showing the distribution of H4R3me2s + cells in cortical organoids at day 50. The average expression of H4R3me2s in each cell is quantified in each vertical position bin (lower left). The normalized abundance of H4R3me2s + cells in each vertical position bin is calculated as the number of H4R3me2s + cells in a bin / the total number of H4R3me2s + cells. CTRL, n=17; OXR1 ΔEx18 , n=14 from two independent batches. f , Immunohistochemical analyses showing the distribution of H3R2me2s + cells of cortical organoids at day 50. The average expression of H4R3me2s of each cell is quantified in each vertical position bin (lower left). The normalized abundance of H3R2me2s+ cells in each vertical position bin is calculated as in ( e ). CTRL, n = 37; OXR1 ΔEx18 , n=38 from two independent batches. g , Immunohistochemical analyses of midbrain organoids at day 20 and day 50 using antibodies specific for FOXA2 (magenta, the marker of midbrain floor plate/progenitor) and TH (yellow, the marker of dopaminergic neurons). h , Immunohistochemical analyses showing Ki67 + proliferating cells (green) in the VZ of midbrain organoids at day 20. The percentage of Ki67 + cells in progenitor zones (VZ) is quantified. CTRL, n=9 organoids; OXR1 ΔEx18 , n=6 organoids. i , Immunohistochemical analyses showing transient expression of EN2 (the marker of mid-hindbrain boundary) in midbrain organoids at day 20. j , Immunohistochemical analyses showing the spatial-temporal distribution of H4R3me2s in midbrain organoids at day 50. The average expression of H4R3me2s in each cell is quantified in each vertical position bin (lower left). The normalized abundance of H4R3me2s + cells in each bin is calculated as in ( e ). CTRL, n = 23; OXR1 ΔEx18 , n=23 from two independent batches. d-f, h-j , Values represent mean ± SEM, * p < 0.05, ** p < 0.01, ***p < 0.001, Student’s t test. In all confocal images, the scale bar is 60 µm and the nuclei were visualized using DAPI (blue).

Article Snippet: The lysate was pre-cleared by Dynabeads™ Protein A (Thermofisher Scientific, Cat#10001D) beads for 30 min. Add 1μg of purified rabbit anti IgG antibody (Sigma-Aldrich Cat# I5006, RRID:AB_1163659) or 3μg OXR1 antibody (Bethyl Cat# A302-036A, RRID:AB_1576565) to the 1mg pre-clear cell lysate separately.

Techniques: Derivative Assay, Generated, Immunohistochemical staining, Expressing, Marker

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) Biotin-labeled ssDNA or complemented dsDNA were conjugated on streptavidin magnetic beads and incubated with GST-tagged FOXP1 purified from E. coli . Streptavidin-bound FOXP1 was detected via immunoblotting with the indicated antibodies. *, degraded GST-FOXP1. ( B ) Biotin-labeled ssDNA conjugated on streptavidin magnetic beads were incubated with GST-tagged FOXP1 or a GST-tagged FOXP1 Δ465–555 mutant purified from E. coli . Streptavidin-bound FOXP1 was detected via immunoblotting with the indicated antibodies. ( C ) Biotin-labeled ssDNA conjugated on streptavidin magnetic beads, with or without preincubation with His-tagged RPA70/RPA32, were incubated with GST-tagged FOXP1. Streptavidin-bound proteins were detected via immunoblotting with the indicated antibodies. ( D , E ) His-tagged RPA32 ( D ) or His-tagged-RPA70 (E) were incubated with GST-tagged FOXP1 before His pull-down assay. Proteins bound onto Ni beads were detected via immunoblotting with the indicated antibodies. ( F ) HEK293T cells treated with 2 mM HU for 1 h or untreated were subjected to immunoprecipitation using a RPA70-specific antibody. The immunoprecipitates were analyzed via immunoblotting with the indicated antibodies. ( G ) Proximity ligation assay experiments using FOXP1 and biotin-specific antibodies in H1975 cells. Lower: representative images of PLA foci. upper: quantification of the number of PLA foci per foci-positive cells (cell number: CON, n = 124; HU-1h, n = 95; HU-2h, n = 94), mean ± SD is shown. **** P < 0.0001, P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: CON vs HU 1 h, 7.20e-014; CON vs HU 2 h, 6.02e-027. ( H ) HEK293T cells were labeled with EdU followed by analysis by iPOND assay and immunoblotting with the indicated antibodies. .

Article Snippet: Rabbit anti-RPA70 polyclonal antibody , Bethyl Laboratories , Cat # A300-241A.

Techniques: Labeling, Magnetic Beads, Incubation, Purification, Western Blot, Mutagenesis, Pull Down Assay, Immunoprecipitation, Proximity Ligation Assay

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: ( A ) Biotin-labeled random dsDNA or dsDNA containing GTAAACA consensus motif was conjugated on streptavidin magnetic beads and incubated with GST-tagged FOXP1 purified from E. coli . Streptavidin-bound FOXP1 was detected via immunoblotting with the indicated antibodies. ( B ) Biotin-labeled random ssDNA or ssDNA containing GTAAACA was used to test FOXP1 binding as described in ( A ). ( C ) Schematic of full-length FOXP1 and its deletion mutants. ( D ) HEK293T cells transfected with FLAG-FOXP1 or its deletion mutants were subjected to immunoprecipitation using a FLAG-specific antibody. FOXP1 bound RPA proteins were detected via immunoblotting. ( E , F ) His-tagged RPA32 ( E ) or RPA70 ( F ) were incubated with GST-tagged FOXP1 (or its deletion mutants) followed by GST pull-down assay. Proteins bound onto Glutathione beads were detected via immunoblotting using the indicated antibodies. ( G ) Proximity ligation assay experiments using FOXP1 and biotin-specific antibodies in H1975 cells transfected with a negative control siRNA or siRNAs targeting RPA32 or RPA70. Upper panel: quantification of the number of PLA foci per foci-positive cells (cell number: siNC CON, n = 103; siRPA70-CON, n = 102; siRPA32-CON, n = 102; siNC HU, n = 110; siRPA70-CON, n = 106; siRPA32-CON, n = 106), mean ± SD is shown. ** P < 0.01, **** P < 0.0001, P values were calculated by one-way ANOVA, followed by Kruskal–Wallis test. P value: siNC CON vs siRPA70-CON, 1.39e-018; siNC CON vs siRPA32-CON, 0.0039; siNC HU vs siRPA70 HU, 5.80e-038; siNC HU vs siRPA32 HU, 1.41e-010. Lower panel: representative images of PLA foci and immunoblotting of RPA expression.

Article Snippet: Rabbit anti-RPA70 polyclonal antibody , Bethyl Laboratories , Cat # A300-241A.

Techniques: Labeling, Magnetic Beads, Incubation, Purification, Western Blot, Binding Assay, Transfection, Immunoprecipitation, Pull Down Assay, Proximity Ligation Assay, Negative Control, Expressing

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-RPA70 polyclonal antibody , Bethyl Laboratories , Cat # A300-241A.

Techniques: Recombinant, Sequencing, shRNA, Modification, Transfection, Protease Inhibitor, Clone Assay, In Situ, Software

Journal: Cell

Article Title: Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription

doi: 10.1016/j.cell.2019.06.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-RBFOX2 , Bethyl , A300-864A; RRID: AB_609476.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Staining, Blocking Assay, Ligation, Purification, Gel Extraction, Imaging, Sequencing, Software

Journal: Cell

Article Title: Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription

doi: 10.1016/j.cell.2019.06.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-SFPQ , Bethyl , A301-321A; RRID: AB_937993.

Techniques: Recombinant, Protease Inhibitor, Magnetic Beads, Staining, Blocking Assay, Ligation, Purification, Gel Extraction, Imaging, Sequencing, Software