Journal: The Journal of Physiology

Article Title: Chronic fetal hypoxia disrupts the peri‐conceptual environment in next‐generation adult female rats

doi: 10.1113/JP277431

Figure Lengend Snippet: A , oviductal telomere length in adult female rats exposed to gestational hypoxia compared to normoxia. B , effect of gestational hypoxia compared to normoxia on gene expression of components ( Ku70 and Ku80 ) of the DNA‐activated protein kinase (DNA‐PK) in the oviducts. C , effect of gestational hypoxia compared to normoxia on protein expression of KU70 and KU80. Data shown as the mean ± SEM. Open bars: normoxia (21% oxygen) during gestation, grey bars: hypoxia (13% oxygen) during gestation. * P < 0.05, *** P < 0.001. n = 7–8 for all groups ( n refers to the number of litters)

Article Snippet: Detection steps used the following primary antibodies: P53 (R&D Systems, R&D Systems, Minneapolis, MN, USA; catalogue no. MAB1355; dilution 1:1000; RRID:AB_357649), P16 INK (Abcam, Cambridge, UK; catalogue no. Ab189034; dilution 1:1000; RRID:AB_2737282), OGG1 (Novus Biologicals, Littleton, CO, USA; catalogue no. NB100‐106; dilution 1:1000; RRID:AB_10104097), MRE11 (ProteinTech, Cambridge, UK; catalogue no. 10744‐1‐AP; dilution 1:1000; RRID:AB2145118), KU70 (ProteinTech; catalogue no. 10723‐1‐AP; dilution 1:1000; RRID:AB_), KU80 (Novus Biologicals; catalogue no. NB100‐508; dilution 1:1000; RRID:AB_2218756), Total Ox Phos rodent antibody cocktail (Abcam; catalogue no. Ab110413; dilution 1:5000; RRID:AB_2629281), HIF1α (Abcam; catalogue no. Ab51608; dilution 1:1000; RRID:AB_880418), GP91 phox (ProteinTech; catalogue no. 19013‐1‐AP; RRID:AB_1342287; dilution 1:1000), P47 phox (ProteinTech; catalogue no. 15551‐1‐AP; dilution 1:1000; RRID:AB_11182937), XO (Santa‐Cruz, Wimbledon, UK; catalogue no. SC‐20991; dilution 1:200, RRID:AB_2214858), HMOX1 (ProteinTech; catalogue no. 20960‐1‐AP; dilution 1:1000; RRID:AB_10732601), Catalase (Abcam; catalogue no. Ab1877‐10; dilution 1:10,000; RRID:AB_187710), MnSOD (Upstate, Watford, UK; catalogue no. 06‐984; RRID:AB_310325; dilution 1:1000), CuZnSOD (ProteinTech; catalogue no. 10269‐1‐AP; dilution 1:1000; RRID:AB_2193750).

Techniques: Gene Expression, Expressing

Journal: The Journal of Physiology

Article Title: Chronic fetal hypoxia disrupts the peri‐conceptual environment in next‐generation adult female rats

doi: 10.1113/JP277431

Figure Lengend Snippet: Effect of gestational hypoxia compared to normoxia on gene expression in the oviducts of adult female rats

Article Snippet: Detection steps used the following primary antibodies: P53 (R&D Systems, R&D Systems, Minneapolis, MN, USA; catalogue no. MAB1355; dilution 1:1000; RRID:AB_357649), P16 INK (Abcam, Cambridge, UK; catalogue no. Ab189034; dilution 1:1000; RRID:AB_2737282), OGG1 (Novus Biologicals, Littleton, CO, USA; catalogue no. NB100‐106; dilution 1:1000; RRID:AB_10104097), MRE11 (ProteinTech, Cambridge, UK; catalogue no. 10744‐1‐AP; dilution 1:1000; RRID:AB2145118), KU70 (ProteinTech; catalogue no. 10723‐1‐AP; dilution 1:1000; RRID:AB_), KU80 (Novus Biologicals; catalogue no. NB100‐508; dilution 1:1000; RRID:AB_2218756), Total Ox Phos rodent antibody cocktail (Abcam; catalogue no. Ab110413; dilution 1:5000; RRID:AB_2629281), HIF1α (Abcam; catalogue no. Ab51608; dilution 1:1000; RRID:AB_880418), GP91 phox (ProteinTech; catalogue no. 19013‐1‐AP; RRID:AB_1342287; dilution 1:1000), P47 phox (ProteinTech; catalogue no. 15551‐1‐AP; dilution 1:1000; RRID:AB_11182937), XO (Santa‐Cruz, Wimbledon, UK; catalogue no. SC‐20991; dilution 1:200, RRID:AB_2214858), HMOX1 (ProteinTech; catalogue no. 20960‐1‐AP; dilution 1:1000; RRID:AB_10732601), Catalase (Abcam; catalogue no. Ab1877‐10; dilution 1:10,000; RRID:AB_187710), MnSOD (Upstate, Watford, UK; catalogue no. 06‐984; RRID:AB_310325; dilution 1:1000), CuZnSOD (ProteinTech; catalogue no. 10269‐1‐AP; dilution 1:1000; RRID:AB_2193750).

Techniques: Gene Expression

Journal: The Journal of Physiology

Article Title: Chronic fetal hypoxia disrupts the peri‐conceptual environment in next‐generation adult female rats

doi: 10.1113/JP277431

Figure Lengend Snippet: Effect of gestational hypoxia compared to normoxia on protein expression in the oviducts of adult female rats

Article Snippet: Detection steps used the following primary antibodies: P53 (R&D Systems, R&D Systems, Minneapolis, MN, USA; catalogue no. MAB1355; dilution 1:1000; RRID:AB_357649), P16 INK (Abcam, Cambridge, UK; catalogue no. Ab189034; dilution 1:1000; RRID:AB_2737282), OGG1 (Novus Biologicals, Littleton, CO, USA; catalogue no. NB100‐106; dilution 1:1000; RRID:AB_10104097), MRE11 (ProteinTech, Cambridge, UK; catalogue no. 10744‐1‐AP; dilution 1:1000; RRID:AB2145118), KU70 (ProteinTech; catalogue no. 10723‐1‐AP; dilution 1:1000; RRID:AB_), KU80 (Novus Biologicals; catalogue no. NB100‐508; dilution 1:1000; RRID:AB_2218756), Total Ox Phos rodent antibody cocktail (Abcam; catalogue no. Ab110413; dilution 1:5000; RRID:AB_2629281), HIF1α (Abcam; catalogue no. Ab51608; dilution 1:1000; RRID:AB_880418), GP91 phox (ProteinTech; catalogue no. 19013‐1‐AP; RRID:AB_1342287; dilution 1:1000), P47 phox (ProteinTech; catalogue no. 15551‐1‐AP; dilution 1:1000; RRID:AB_11182937), XO (Santa‐Cruz, Wimbledon, UK; catalogue no. SC‐20991; dilution 1:200, RRID:AB_2214858), HMOX1 (ProteinTech; catalogue no. 20960‐1‐AP; dilution 1:1000; RRID:AB_10732601), Catalase (Abcam; catalogue no. Ab1877‐10; dilution 1:10,000; RRID:AB_187710), MnSOD (Upstate, Watford, UK; catalogue no. 06‐984; RRID:AB_310325; dilution 1:1000), CuZnSOD (ProteinTech; catalogue no. 10269‐1‐AP; dilution 1:1000; RRID:AB_2193750).

Techniques: Expressing

Journal: Viruses

Article Title: Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus

doi: 10.3390/v14040755

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The primary antibodies used in this study were specific for XRCC5 (16389-1-AP, Proteintech, Rosemont, IL, USA), XRCC6 (10723-1-AP, Proteintech, Rosemont, IL, USA), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), VP5 (prepared in our lab), and gB (prepared in our lab).

Techniques: Sequencing

Journal: Viruses

Article Title: Proteomic Analysis of Vero Cells Infected with Pseudorabies Virus

doi: 10.3390/v14040755

Figure Lengend Snippet: Validation of proteomics data by western blot and RT-qPCR. ( A ) Vero cells infected with PRV for 24 h were collected and western blot was performed to detect the expression of XRCC5 and XRCC6 with corresponding antibodies. ( B ) The western blot and proteomics ratio of XRCC5 and XRCC6. ( C ) Relative XRCC6 transcription in Vero cells. ( D ) Relative XRCC5 transcription in Vero cells. ( E ) The expression of XRCC5 and XRCC6 in PK-15 infected with PRV. ( F ) The expression of XRCC5 and XRCC6 in CRL-2843 infected with PRV. ** indicates significance at a 99% confidence interval ( p < 0.01).

Article Snippet: The primary antibodies used in this study were specific for XRCC5 (16389-1-AP, Proteintech, Rosemont, IL, USA), XRCC6 (10723-1-AP, Proteintech, Rosemont, IL, USA), β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA), VP5 (prepared in our lab), and gB (prepared in our lab).

Techniques: Biomarker Discovery, Western Blot, Quantitative RT-PCR, Infection, Expressing

Journal: EBioMedicine

Article Title: The BET inhibitor JQ1 attenuates double-strand break repair and sensitizes models of pancreatic ductal adenocarcinoma to PARP inhibitors

doi: 10.1016/j.ebiom.2019.05.035

Figure Lengend Snippet: JQ1 decreased the expression of Ku80 and RAD51. (a) Immunohistochemistry (IHC) was done to detect the expression of Ku80 or RAD51 protein in normal pancreas or primary PDAC tumors. Primary PDAC tumors, UAB-PA4 and UAB-PA16, expressed higher levels of the NHEJ protein Ku80 and HR protein RAD51 compared to a normal pancreas (NP2). Quantitation of IHC results is shown as expression indices (were calculated as described in Materials and Methods) in the lower left-hand corner of each photomicrograph. Scale bar = 10 μM. (b) Forty-eight hour treatment of JQ1 (10 μM) decreased mRNA levels of both Ku80 and RAD51 in BxPC3 or Panc1 PDAC cells using qRT-PCR assays. The sequences for primers used are in Table S1 (Supplementary Materials). Data is shown as the mean ± S.E.M. Two-way analysis of variance (ANOVA) was performed (* p < .05, ** p < .01) using Prism. (c) Immunoblot demonstrating that JQ1 (0.5, 1, 5, or 10 μM) decreased protein expression of Ku80 and RAD51 in BxPC3 or Panc1 PDAC cells treated for 48 h. (d) The immunoblot data in c were quantitated as percent DMSO using ImageStudio Lite (LI-COR Biosciences) and are reported as bar graphs mean ± S.D. Analysis was done by two-way ANOVA (**** p < .0001).

Article Snippet: Primary antibodies used were: γH2AX (Cell Signaling), RAD51 (abcam, Cambridge, MA, USA), Ku80 (Bethyl Laboratories, Montgomery, TX, USA), BRD4 (Cell Signaling), BRD2 (Cell Signaling), vinculin (Santa Cruz Biotech, Dallas, TX, USA), GAPDH (Cell Signaling), β-actin (Cell Signaling), α-Tubulin (Cell Signaling), p21 (Cell Signaling), Cleaved PARP (Cell Signaling).

Techniques: Expressing, Immunohistochemistry, Quantitation Assay, Quantitative RT-PCR, Western Blot

Journal: EBioMedicine

Article Title: The BET inhibitor JQ1 attenuates double-strand break repair and sensitizes models of pancreatic ductal adenocarcinoma to PARP inhibitors

doi: 10.1016/j.ebiom.2019.05.035

Figure Lengend Snippet: JQ1 + olaparib increased the levels of DNA damage marker γH2AX and reduced the expression of Ku80 and RAD51 in in vivo models of PDAC. Immunoblots (IB) were done to detect γH2AX and DNA repair proteins, Ku80 and RAD51 using UAB-PA4 (a) or UAB-PA16 (b) tumors harvested from mice 24 h following final treatment (see Materials and Methods). Quantitation by densitometry of results is shown below each IB image. Immunohistochemistry was done to detect γH2AX, apoptosis marker cleaved caspase 3 (cl. casp 3) and DNA repair proteins, Ku80 and RAD51 using UAB-PA4 (c) or UAB-PA16 (d) thin sections of tumors harvested from mice 24 h following final treatment. Scale bar = 10 μM. Quantitation of results is shown either as a bar graph for γH2AX and cleaved caspase 3 (cl. casp 3) (e, f for UAB-PA4 and UAB-PA16, respectively) or as expression indices (were calculated as described in Materials and Methods) in the lower left-hand corner of each photomicrograph depicting IHC image. Data are presented as mean ± S.E.M. and analyzed with one-way ANOVA (* p < .05, ** p < .01, *** p < .001, **** p < .0001).

Article Snippet: Primary antibodies used were: γH2AX (Cell Signaling), RAD51 (abcam, Cambridge, MA, USA), Ku80 (Bethyl Laboratories, Montgomery, TX, USA), BRD4 (Cell Signaling), BRD2 (Cell Signaling), vinculin (Santa Cruz Biotech, Dallas, TX, USA), GAPDH (Cell Signaling), β-actin (Cell Signaling), α-Tubulin (Cell Signaling), p21 (Cell Signaling), Cleaved PARP (Cell Signaling).

Techniques: Marker, Expressing, In Vivo, Western Blot, Quantitation Assay, Immunohistochemistry

Journal: EBioMedicine

Article Title: The BET inhibitor JQ1 attenuates double-strand break repair and sensitizes models of pancreatic ductal adenocarcinoma to PARP inhibitors

doi: 10.1016/j.ebiom.2019.05.035

Figure Lengend Snippet: Simultaneous exposure of JQ1 + PARPi increased DNA damage, reduced expression of DNA repair proteins Ku80 and RAD51, inhibited colony formation, and induced synergistic cytotoxicity in PDAC cells. BxPC3 (a) or Panc1 (b) cells were exposed to the indicated concentrations of JQ1 (0.1-100 μM) in combination with 20 μM veliparib or 10 μM olaparib for 72 h, after which alamarBlue solution was added and fluorescence read. Data were normalized to controls at each time point, with control values = 100%. Each point represents the average of quadruplicate wells from three independent assays. Data are presented as mean ± S.D. IC 50 values of JQ1 as a single agent or in combination with veliparib or olaparib are shown in a table below the graph. (c) JQ1 + veliparib increased DNA damage, as reflected by an increase in γH2AX positive BxPC3 cells. The percent of γH2AX-positive cells following exposure to DMSO, veliparib (10 μM), JQ1 (3.5 μM), or JQ1 (3.5 μM) + veliparib (10 μM) for 24 h were quantitated. The % of γH2AX positive cells was calculated by counting the number of positive cells (cells containing ≥5 foci) and dividing it by the total number of cells in two independent experiments (a minimum of 200 cells were counted per experiment). A representative IF stain is shown for each treatment group. Data presented as mean ± S.E.M. and analyzed by one-way ANOVA (** p < .01, *** p < .001). (d) Panc1 cells were exposed to DMSO, JQ1 (1 μM), olaparib (1 μM) or the combination (1:1) for 72 h, at which time drug-containing medium was replaced with drug-free medium and the cells were cultured for an additional 11 days. On day 14, cells were fixed and stained with crystal violet, and quantitation of viable colonies done by determining number of colonies having >50 cells. The data are shown as a box and whisker plot with the whiskers showing the min and max. Analysis was done using a one-way ANOVA (* p < .05, ** p < .01). (e) JQ1 + PARPi are synergistic in PDAC cells. BxPC3 and Panc1 cells were exposed to JQ1 + veliparib (1:1) (0.1-25 μM) or JQ1 + olaparib (1:1) (0.1-100 μM) for 72 h, and cell viability analyzed by alamarBlue assay. Combination indices (CI) are plotted to fraction of cells affected (Fa) using CompuSyn software which was based on the Chou-Talalay method. A CI of <1.0 indicates synergism. (f) JQ1 and JQ1 + olaparib increased the levels of γH2AX and p21, and decreased the expression of Ku80 and RAD51 in BxPC3 cells. BxPC3 cells were exposed to the indicated concentrations of JQ1 (0.1, 1, and 10 μM) with or without 10 μM olaparib for 48 h, and cell lysates were immunostained for the indicated proteins. (g) RAD51 nuclear foci formation assays were done with BxPC3 cells using JQ1 or olaparib as a control. Cells were exposed to JQ1 (20 μM) or olaparib (1 μM) for 24 h, fixed and stained with FITC-RAD51 antibody. DAPI was used to detect nuclei. Quantitation was done by counting the percent (%) RAD51 foci positive cells and plotted as mean ± S.E.M. The % of RAD51 positive cells was calculated by counting the number of positive cells (cells containing ≥5 foci) and dividing it by the total number of cells in three independent experiments (a minimum of 50 cells were counted per experiment). Student's t -test was performed (** p < .01, Scale bar = 10 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used were: γH2AX (Cell Signaling), RAD51 (abcam, Cambridge, MA, USA), Ku80 (Bethyl Laboratories, Montgomery, TX, USA), BRD4 (Cell Signaling), BRD2 (Cell Signaling), vinculin (Santa Cruz Biotech, Dallas, TX, USA), GAPDH (Cell Signaling), β-actin (Cell Signaling), α-Tubulin (Cell Signaling), p21 (Cell Signaling), Cleaved PARP (Cell Signaling).

Techniques: Expressing, Fluorescence, Control, Staining, Cell Culture, Quantitation Assay, Whisker Assay, Alamar Blue Assay, Software

Journal: EBioMedicine

Article Title: The BET inhibitor JQ1 attenuates double-strand break repair and sensitizes models of pancreatic ductal adenocarcinoma to PARP inhibitors

doi: 10.1016/j.ebiom.2019.05.035

Figure Lengend Snippet: Expression of Ku80 and RAD51 was BET-dependent. Chromatin immunoprecipitation assays of BRD4 in BxPC3 cells (a) or BRD2 in Panc1 cells (b) on the promoter sequences of Ku80 and RAD51. Forty-eight hour exposure to JQ1 (10 μM) decreased the association of BRD4 or BRD2 with the promoter sequences of Ku80 and RAD51 compared to the vehicle (DMSO) controls. Rabbit IgG was used as a negative control. The data is shown as the mean ± S.D. and two-way ANOVA was used to identify differences in levels of expression (* p < .05, *** p < .001). Experiments in which shRNA was used to decrease expression of BRD4 in BxPC3 (c) or BRD2 (d) in Panc1 cells using shRNA (see Table S1 for sequences), decreased the expression of both Ku80 and RAD51. Quantitation of 6c and 6d is shown in the right panel as the mean ± S.D. in the bar graph. Two-way ANOVA was used to determine significance (**** p < .0001). shGFP serves as a control.

Article Snippet: Primary antibodies used were: γH2AX (Cell Signaling), RAD51 (abcam, Cambridge, MA, USA), Ku80 (Bethyl Laboratories, Montgomery, TX, USA), BRD4 (Cell Signaling), BRD2 (Cell Signaling), vinculin (Santa Cruz Biotech, Dallas, TX, USA), GAPDH (Cell Signaling), β-actin (Cell Signaling), α-Tubulin (Cell Signaling), p21 (Cell Signaling), Cleaved PARP (Cell Signaling).

Techniques: Expressing, Chromatin Immunoprecipitation, Negative Control, shRNA, Quantitation Assay, Control

Journal: bioRxiv

Article Title: Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCF β TRCP -dependent ubiquitylation of the actin-organizing protein Dbn1

doi: 10.1101/2023.05.15.540799

Figure Lengend Snippet: A. Experimental outline for the UBIMAX experiment profiling ubiquitylated proteins in response to DPC-containing substrates. Egg extracts were left untreated or supplemented with ubiquitin E1 inhibitor (“Ub E1i”) prior to addition of His 6 -Ubiquitin (“His 6 -Ub”). Reactions were initiated by addition of buffer (“no DNA”), undamaged plasmid DNA (“DNA”), plasmids carrying the M.HpaII protein crosslinked at a single-stranded DNA gap (“ssDNA-DPC”), or plasmids carrying the Flp protein crosslinked at a single-strand break (“SSB-DPC”). Reactions were performed in triplicate from the same batch of egg extracts. Samples were transferred to denaturing pulldown buffer 30 min after initiation of the reaction and subjected to the UBIMAX workflow as outlined in . B-C. Volcano plot analysis comparing ubiquitylated proteins enriched from ssDNA-DPC ( B ) or SSB-DPC ( C ) versus DNA-treated samples. Pink/orange and blue dots indicate significantly enriched and depleted ubiquitylated proteins, respectively. Significance was determined by two-tailed Student’s t test, with permutation-based FDR-control with S0 = 0.1 and 2500 rounds of randomization, to ensure an FDR ≤ ss0.05. Ubiquitylated proteins with FDR ≤ 0.01 are labelled. N=3. D-I. Abundance distributions of Ku80 ( D ), Ku70 ( E ), Mre11 ( F ), Rpa1 ( G ), Chfr ( H ) and Dbn1 ( I ) across the ubiquitin target enriched samples of the UBIMAX experiments profiling protein ubiquitylation in response to DSBs and DPCs (A), respectively. Horizontal lines indicate the median and significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test for all conditions against undamaged DNA with a cut-off of p-value ≤ 0.01. N=3-7. a.u., arbitrary units. J. Egg extracts were left untreated or supplemented with ubiquitin E1 inhibitor prior to initiation of the reactions by addition of either undamaged plasmid DNA (“DNA”), linearized plasmid DNA (“DSB”), or plasmids carrying a DPC at a ssDNA gap (“ssDNA-DPC”). Samples were transferred to sample buffer at the indicated times and analysed by WB using antibodies against Dbn1 and Ku80. Mcm6 served as a loading control. * denotes an unspecific band.

Article Snippet: The following antibodies were raised against the indicated peptides derived from Xenopus laevis proteins (New England Peptide now Biosynth): Dbn1 (Ac-CWDSDPVMEEEEEEEEGGGFGESA-OH), Ku80 (CMEDEGDVDDLLDMM), Cul1 (H2N-MSSNRSQNPHGLKQIGLDQC-amide), Fbxl12 (Ac-CRGIDELKKSLPNSKVTN-OH), Psa3 (Ac-CKYAKESLEEEDDSDDDNM-OH), β-Trcp1-INT (Ac-GQYLFKNKPPDGKTPPNSC-amide), β-Trcp1-NT (H2N-MEGFSSSLQPPTASEREDC-amide), and Dbn1-pS609/611 (Ac-CSEGYF(pS)Q(pS)QDED-amide).

Techniques: Ubiquitin Proteomics, Plasmid Preparation, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCF β TRCP -dependent ubiquitylation of the actin-organizing protein Dbn1

doi: 10.1101/2023.05.15.540799

Figure Lengend Snippet: A. Dbn1 immunodepletion control of the experiment shown in . An egg extract dilution range is shown for comparison. B. Egg extracts were left untreated or supplemented with ATM or ATR inhibitor (“ATMi”, “ATRi”) prior to addition of linearized plasmid DNA (“DSB”). Samples were transferred to sample buffer at the indicated timepoints and analysed by WB using antibodies against Dbn1 and Chk1-pS345. Orc2 served as a loading control. C. His 6 -Ubiquitin (“His 6 -Ub”) WT or the indicated mutants were added to egg extracts at a final concentration of 1 µg/µl. Reactions were initiated by addition of linearized plasmid DNA, samples collected after 30 minutes, and subjected to denaturing His-ubiquitin pulldown followed by WB analysis using antibodies against Dbn1. PD, pulldown. D. Egg extracts were left untreated or supplemented with proteasome inhibitor (MG262) and His 6 -ubiquitin prior to addition of linearized plasmid DNA. Samples were collected at the indicated timepoints and subjected to denaturing His-ubiquitin pulldown followed by WB analysis using antibodies against Dbn1. Ubiquitin served as a pulldown control. E. Linearized plasmid DNA was added to Mock-, Cul1-or Fbxl12-immunodepleted egg extracts and samples transferred to sample buffer at the indicated timepoints. Samples were analysed by WB using antibodies against Dbn1 and Ku80 (long and short exposures shown). Mcm6 served as a loading control. Cul1-and Fbxl12 immunodepletion controls are shown to the right. Depl. rnd, immunodepletion round. * denotes an unspecific band. F. Abundance distribution of the two Xenopus laevis Dbn1 isoforms, Dbn1.S and Dbn1.L, across the sample groups of the Dbn1 IP-MS experiment outlined in . Horizontal lines indicate the median. N=4. a.u., arbitrary units. G. Unstimulated egg extracts were subjected to immunoprecipitation using IgG-(“mock”), Cul1-, β-Trcp1-INT, or β-Trcp1-NT antibodies, followed by MS analysis. Shown are the abundance distributions of Skp1, Cul1, and β-Trcp1. Horizontal lines indicate the median and significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test for all immunoprecipitation conditions compared to the mock control. N=3. a.u., arbitrary units. H. Linearized plasmid DNA was added to mock-or β-Trcp1-immunodepleted egg extracts (using either β-Trcp1-INT or β-Trcp1-NT antibodies) and samples transferred to sample buffer at the indicated timepoints. Samples were analysed by WB using antibodies against Dbn1 (long and short exposures shown). Mcm6 served as a loading control. I. His 6 -ubiquitin and linearized plasmid DNA were added to mock-, Cul1-, or β-Trcp1-immunodepleted egg extracts. Samples were collected from immunodepleted egg extract prior to addition of DNA (“input”), and 60 minutes after addition of linearized DNA for denaturing His-ubiquitin pulldown. Samples were analysed by WB using antibodies against Dbn1. J. Recombinant β-Trcp1 protein or buffer was added to mock- and β-Trcp1-immunodepleted egg extracts as indicated prior to addition of His 6 -ubiquitin and linearized plasmid DNA. Input and pulldown samples were collected 60 minutes after addition of linearized plasmid DNA and processed as described in (J). K. HeLa cells were transfected with control or siRNA targeting DBN1 before being subjected or not to 10 Gy ionizing radiation (IR). Lysates were harvested 30 minutes after irradiation, subjected to ubiquitin pulldown, and analysed along with whole cell extracts (“input”) by WB using antibodies against DBN1. GAPDH served as a loading control. PD, pulldown; Ub, ubiquitin.

Article Snippet: The following antibodies were raised against the indicated peptides derived from Xenopus laevis proteins (New England Peptide now Biosynth): Dbn1 (Ac-CWDSDPVMEEEEEEEEGGGFGESA-OH), Ku80 (CMEDEGDVDDLLDMM), Cul1 (H2N-MSSNRSQNPHGLKQIGLDQC-amide), Fbxl12 (Ac-CRGIDELKKSLPNSKVTN-OH), Psa3 (Ac-CKYAKESLEEEDDSDDDNM-OH), β-Trcp1-INT (Ac-GQYLFKNKPPDGKTPPNSC-amide), β-Trcp1-NT (H2N-MEGFSSSLQPPTASEREDC-amide), and Dbn1-pS609/611 (Ac-CSEGYF(pS)Q(pS)QDED-amide).

Techniques: Immunodepletion, Control, Comparison, Plasmid Preparation, Ubiquitin Proteomics, Concentration Assay, Protein-Protein interactions, Immunoprecipitation, Recombinant, Transfection, Irradiation

Journal: bioRxiv

Article Title: Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCF β TRCP -dependent ubiquitylation of the actin-organizing protein Dbn1

doi: 10.1101/2023.05.15.540799

Figure Lengend Snippet: A. Mock- or Dbn1-immunodepleted egg extracts were supplemented with His 6 -ubiquitin (“His 6 -Ub”) prior to addition of linearized plasmid DNA (“DSB”). Samples were collected at the indicated times and subjected to denaturing His-ubiquitin pulldown followed by WB analysis using antibodies against Dbn1. Ubiquitin served as a pulldown control. Immunodepletion control is provided in . PD, pulldown. B. Egg extracts were left untreated or supplemented with ATM inhibitor (“ATMi”) prior to addition of linearized plasmid DNA. Samples were transferred to sample buffer at the indicated timepoints and analysed by WB using antibodies against Dbn1 (long and short exposures shown) and Chk1-pS345. Orc2 served as a loading control. C. Egg extracts were left untreated or supplemented with neddylation E1 inhibitor (“Culi”) prior to addition of linearized plasmid DNA. Samples were transferred to sample buffer at the indicated timepoints and analysed by WB using antibodies against Dbn1. Orc2 served as a loading control. D. Recombinant dominant negative Cullin proteins or buffer was mixed with egg extracts prior to addition of linearized plasmid DNA. Samples were transferred to sample buffer at the indicated timepoints and analysed by WB using antibodies against Dbn1, Ku80 (long and short exposures shown) and Cdt1. Mcm6 served as a loading control. * denotes an unspecific band. E. Experimental outline of Dbn1 IP-MS experiment. Egg extracts were left untreated or supplemented with ATM inhibitor or proteasome inhibitor (MG262) prior to addition of undamaged-(“DNA”) or linearized plasmid DNA (“DSB”) as indicated. Reactions were performed in quadruplicate from the same batch of egg extracts. Samples were collected at 60 min, subjected to mock-or Dbn1-immunoprecipitation as indicated and analysed by MS. F. Volcano plot analysis comparing the proteins enriched from Dbn1 IP-MS samples treated with DSB with versus without proteasomal inhibition. Orange and blue dots indicate significantly enriched and depleted ubiquitylated proteins, respectively. Significance was determined by two-tailed Student’s t test, with permutation-based FDR-control with S0 = 0.1 and 2500 rounds of randomization, to ensure an FDR ≤ 0.05. N=4. G. Scatter plot analysis of the Dbn1 IP-MS experiment detailed in (F). The mean difference in abundance between proteins enriched in linearized-and undamaged plasmid DNA-treated samples is plotted against that of linearized DNA without versus with ATM inhibition. Red and blue dots indicate proteins significantly enriched and depleted with Dbn1 immunoprecipitation in the presence of linearized plasmid DNA, respectively. Purple dots or outlines indicate proteins significantly changed in enrichment with Dbn1 immunoprecipitation upon ATM inhibition. Significance was determined by two-tailed Student’s t test with S0 = 0.1 and FDR ≤ 0.05. N=4. H. Abundance distributions of Skp1, Cul1, Nedd8, and β-Trcp1 across the indicated Dbn1 IP-MS conditions. Horizontal lines indicate the median and significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test for all conditions shown against linearized plasmid DNA. N=4. a.u., arbitrary units. I. Recombinant β-Trcp1 protein or buffer was mixed with mock-or β-Trcp1-immunodepleted egg extracts as indicated prior to addition of linearized plasmid DNA. Samples were transferred to sample buffer at the indicated timepoints and analysed by WB using antibodies against Dbn1 (long and short exposures shown). Psa3 served as a loading control. J. HeLa cells were subjected to 10 Gy ionizing radiation (IR) and harvested after the indicated timepoints. Lysates were subjected to ubiquitin pulldown and analysed along with whole cell extracts (“input”) by WB using antibodies against DBN1. PD, pulldown; Ub, ubiquitin. K. HeLa cells were left untreated (“Unt.”) or treated with neddylation E1 inhibitor (“Culi”) or ATM inhibitor (“ATMi”) for 1 hour before being subjected or not to 10 Gy IR. Cells were harvested 30 minutes after irradiation and processed as described in (J). WB analysis of CUL4A served as a control for the neddylation E1 inhibitor. L. HeLa cells were transfected with control siRNA or two different siRNAs targeting β-Trcp1 for 72h before being subjected or not to 10 Gy IR. Cells were harvested 30 minutes after irradiation and processed as described in (J).

Article Snippet: The following antibodies were raised against the indicated peptides derived from Xenopus laevis proteins (New England Peptide now Biosynth): Dbn1 (Ac-CWDSDPVMEEEEEEEEGGGFGESA-OH), Ku80 (CMEDEGDVDDLLDMM), Cul1 (H2N-MSSNRSQNPHGLKQIGLDQC-amide), Fbxl12 (Ac-CRGIDELKKSLPNSKVTN-OH), Psa3 (Ac-CKYAKESLEEEDDSDDDNM-OH), β-Trcp1-INT (Ac-GQYLFKNKPPDGKTPPNSC-amide), β-Trcp1-NT (H2N-MEGFSSSLQPPTASEREDC-amide), and Dbn1-pS609/611 (Ac-CSEGYF(pS)Q(pS)QDED-amide).

Techniques: Ubiquitin Proteomics, Plasmid Preparation, Control, Immunodepletion, Recombinant, Dominant Negative Mutation, Protein-Protein interactions, Immunoprecipitation, Inhibition, Two Tailed Test, Irradiation, Transfection

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Quercetin inhibits DNA damage responses to induce apoptosis via SIRT5/PI3K/AKT pathway in non-small cell lung cancer.

doi: 10.1016/j.biopha.2023.115071

Figure Lengend Snippet: Fig. 5. Quercetin inhibited NHEJ and HR pathways phosphorylation. Total proteins lysis and extraction after A549 and H1299 cultured with 0, 12.5, 50, and 200 μM quercetin for 24 h. In NHEJ pathways (A) the expression of p-DNA-PKcsS2056 (B, C), KU70 (D, E) and KU80 (F, G), and in HR pathways (H) the phosphorylation of p- ATRS428 (I, J), p-Chek1S345 (K, L), p-ATMS1981 (M, N) and Chek2T68 (O, P) were detected by western blot in both A549 and H1299 cells. And the results were measured by ImageJ and expressed as protein expression relative to GAPDH (mean ± S.D., n = 3). #p > 0.05, *p < 0.05, * *p < 0.01, * **p < 0.001 relative to values in the respective 0 μM group (B, p = 0.0048, R2 =0.7844, F=9.700; C, p = 0.0002, R2 =0.9042, F=25.17; D, p = 0.0054, R2 =0.7782, F=9.358; E, p = 0.0045, R2 =0.7879, F=9.908; F, p = 0.0037, R2 =0.7990, F=10.60; G, p = 0.0016, R2 =0.8379, F=13.79; I, p = 0.0034, R2 =0.8028, F=10.85; J, p = 0.0032, R2 =0.8066, F=11.12; K, p = 0.0010, R2 =0.8564, F=15.91; L, p = 0.0016, R2 =0.8379, F=13.79; M, p = 0.0044, R2 =0.7898, F=10.02; N, p = 0.0065, R2 =0.7676, F=8.806; O, p = 0.0030, R2 =0.0.8087, F=11.27; P, p = 0.0026, R2 =0.8166, F=11.87), One-way ANOVA test. The mRNA in A549 (Q) and H1299 (R) cells was extracted and reverse transcribed to cDNA for RT-qPCR analysis (mean ± S.D., n = 3). #p > 0.05, *p < 0.05, * *p < 0.01, * **p < 0.001, * ** *p < 0.0001 relative to values in the respective 0 μM group, One-way ANOVA test.

Article Snippet: Antibodies to γ-H2AXS139, p-ATRS428 (AP0676) and p-CDK1T161 (AP0324) were from Abclonal Technology (Abclonal, Wuhan, China); antibodies to KU70 (AF0300) and p-Chek1Ser345 (AF3008) were from Affinity Biosciences (Affinity, Jiangsu, China); antibodies to KU80 (PB9464) and p-DNAPKcsS2056 (BM4058) were from Boster Biological Technology (Boster, Wuhan, China); antibodies to p-ATMSER1981 (bsm-54103R) and pChek2Thr68 (ba-3721R) were from Biosynthesis Biotechnology (Bioss, Beijing, China); antibodies to p-PI3K (17366) and p-AKT (4060) were from Cell Signaling Technology (Danvers, MA, USA); antibodies to Caspase-3 (ab32042), Bax (ab32503), Bcl-2 (ab32124), SIRT5 (ab259967), GAPDH (ab9485) and β-tubulin (ab179511) were from Abcam (Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Lysis, Extraction, Cell Culture, Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Bioengineered Skin from a Platelet-Derived Hydrogel Repairs Full Thickness Wounds in a Pre-Clinical Mouse Model

doi: 10.3390/ijms26209988

Figure Lengend Snippet: Full thickness wound repair by engineered skin grafting in athymic nude mice . ( A ) Representative images of the wounds grafted with PG-HSE, BTM-HSE, or native skin autografts on day of grafting (day 0) and post-grafting (day 14). ( B ) Representative microscopic images of day 14 grafts analysed by immunofluorescence, using a human specific antibody against involucrin. PG-HSE pre-grafting (scale 100 µm); PG-HSE graft (scale 1 mm); BTM-HSE pre-grafting (scale 100 µm); BTM-HSE graft (scale 1 mm); autograft pre-grafting (scale 100 µm; autograft (scale 1 mm); and human native skin (scale 1 mm). Zoomed images scale bar = 50 µm. Orange arrows indicate the graft edge 2 weeks post-grafting and red arrows indicate the initial wound edge. ( C ) Presence of human involucrin in grafts is presented as integrated density (IntDen), that is, sum of pixel value/signal intensity within a selected region of interest calculated using FIJI software version 1.54p. ( D ) Human-specific Ku80 marker was detected by immunofluorescence on day 14 post-grafting and % Ku80 positive cells were counted using Nikon NIS-Elements Analysis software (version 5.5). Data were analysed using ordinary one-way ANOVA. Values in ( C , D ) represent mean values +/− SEM in each group (* = p ≤ 0.05, ** = p ≤ 0.01, n = 4–5 per group). PG-HSE, platelet-derived human skin equivalent; BTM-HSE, NovoSorb biodegradable temporising matrix human skin equivalent; autograft, and autologous full thickness skin graft.

Article Snippet: Slides were blocked as above and incubated with primary antibodies: rabbit anti-human involucrin (1:100, Cat. No. ab234403, Abcam), rabbit anti-human Ku80 (1:50, Cat. No. 214745, Biorbyt, Durham, NC, USA), or rabbit anti-human CK5 (1:250, Cat. No. 905504, BioLegend, San Diego, CA, USA ) overnight.

Techniques: Immunofluorescence, Software, Marker, Derivative Assay