bard1 antibody  (Bio-Techne corporation)

Journal: Reproductive Sciences

Article Title: Conservative Hypomethylation of Mesenchymal Stem Cells and Their Secretome Restored the Follicular Development in Cisplatin-Induced Premature Ovarian Failure Mice

doi: 10.1007/s43032-023-01389-4

Figure Lengend Snippet: Phenotype characterization of partially hypomethylated MSCs. The expression of CD105, CD90, CD45, and CD34 was analyzed by flow cytometry for normally methylated cells ( A ), cells treated with 2.5 μM ( B ), or 5 μM ( C ) of 5-Aza-dC. No significant differences were observed in the expression of CD markers between DMSO-treated cells and cells treated with 5-Aza-dC ( D ). Bars represent the average (±SD) of three independent measurements, ns , not significant. Comparison between means was performed by one-way ANOVA test

Article Snippet: Phycoerthrin (PE)-conjugated mouse monoclonal antibodies of CD90, and CD34 or FITC-conjugated monoclonal antibodies of CD105 and CD45 were from (Biotechne R&D System, MN, USA).

Techniques: Expressing, Flow Cytometry, Methylation, Comparison

Journal: Cell Discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: a Incubation of recombinant BRCA1 BRCT or BARD1 BRCT with meiotic spreads. γH2AX is a surrogate marker of the XY body. SyCP3 is a surrogate marker of the unsynapsed axis of X and Y chromosomes. Colocalization of the BRCA1 BRCT and BARD1 BRCT with SyCP3 on the unsynapsed axis was examined. Fluorescence intensity is analyzed on the dashed line. b Meiotic spreads were pre-treated with RNase A prior to the incubation with the recombinant BRCA1 BRCT or BARD1 BRCT. Colocalization of the BRCA1 BRCT and BARD1 BRCT with SyCP3 on the unsynapsed axis was examined. Fluorescence intensity is analyzed. c Incubation IR-treated U2OS cells with recombinant BRCA1 BRCT or BARD1 BRCT. U2OS cells were treated with or without 10 Gy of IR. After a 12-h recovery, the cells were incubated with the recombinant proteins. Recombinant proteins were labeled with anti-GST antibodies. DSBs were labeled with anti-γH2AX antibody. The fluorescence signals on dash lines were analyzed. The percentage of foci-positive cells (> 15 foci) was measured. d Following RNase A or RNase H treatment, the recombinant BRCA1 BRCT- or BARD1 BRCT- recognized IRIF was examined in U2OS cells. Foci number per cell was analyzed. e The IRIF of endogenous BRCA1 and BARD1 was examined in U2OS cells. Foci number per cell was analyzed. P values were calculated using Student’s t -test. n.s. nonsignificant, * P < 0.05, *** P < 0.001. Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Incubation, Recombinant, Marker, Fluorescence, Labeling

Journal: Cell Discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: a Schematic diagram for identification of the BRCA1/BARD1 complex-associated RNA species. b Pie charts showing the BRCA1/BARD1 complex-associated RNA species. c Interactions between pre-rRNA and the BRCA1/BARD1 complex are validated by PAR-CLIP and RT-qPCR. d , e Interactions between the recombinant BRAC1-BRCT and BARD1 BRCT and pre-rRNA were examined by GST pull-down assays. GST, GST-BRCA1 BRCT, or GST-BARD1 BRCT proteins were incubated with pre-rRNA. Following GST pull-down assays, RT-qPCR was performed to examine the enrichment of pre-rRNA. Values are means ± SD of three independent assays. f The BRCT domains of BRCA1 and BARD1 directly interact with rRNA. Binding affinities between the BRCT domains and biotin-labeled RNA oligos (25nt) were measured by BLI assays.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Quantitative RT-PCR, Recombinant, Incubation, Binding Assay, Labeling

Journal: Cell Discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: a A diagram illustrating methods for a mass spectrometry proteomics experiment for the identification of the BRCA1- and BARD1-associated proteins. 293T stably expressing SFB–BRCA1 BRCT or SFB–BARD1 were used for the analyses. SFB empty vector (EV) was used as a negative control. b Pie charts depicting the binding partners of BRCA1 and BARD1 from mass spectrometry analysis. c A myriad of ribosomal proteins identified from the mass spectrometry analysis are shown. BRCA1 BRCT or BARD1-associated large subunit (LSU) and small subunit (SSU) proteins are shown separately. d The BRCA1 BRCT interacts with RPL7A and RPL14. 293T cells stably expressing BRCA1 BRCT were examined. Co-IP and western blotting assay were performed with indicated antibodies. e IR induces interactions between the BRCA1/BARD1 complex and ribosomal proteins. 293 T cells stably expressing BRCA1 BRCT or BARD1 were treated with 10 Gy of IR. Following the indicated recovering time, RPL7A and RPL14 were assessed by co-IP and western blotting assay. The BRCA1 BRCT- or BARD1-associated RPL7A or RPL14 are analyzed in the histograms (bottom panel). Values are means ± SD of three independent assays. P values were calculated using Student’s t -test. n.s. nonsignificant, * P < 0.05, *** P < 0.001. f Associations between the endogenous BRCA1/BARD1 complex and ribosomal proteins were examined by co-IP and western blotting assay with indicated antibodies. g The RNase A treatment abolishes the interactions between the BRCA1/BARD1 complex and ribosomal proteins. The cell lysates isolated from 293T cells stably expressing SFB–BRCA1 BRCT or SFB–BARD1 were treated with or without RNase A prior to IP. The associated RPL7A or RPL14 is summarized in the histograms (bottom panel). Values are means ± SD of three independent assays.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Plasmid Preparation, Negative Control, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Isolation

Journal: Cell Discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: a Pre-rRNP exists at IRIF. Cells were treated with 10 Gy of IR. Pre-rRNA was examined by RNA probes. Ribosomal proteins were stained with indicated antibodies. The colocalization with BRCA1 was analyzed (bottom panel). b Foci number per cell and foci colocalization ratio are examined. c The IRIF of the BRCA1/BARD1 complex in the pol Ii-treated cells. HeLa cells were treated with RNA polymerase inhibitors prior to 10 Gy of IR. The IRIF of BRCA1 and BARD1 were stained with indicated antibodies. Foci number per cell is shown (right panel). The cells were also counterstained by DAPI. P values were calculated using Student’s t -test. Scale bars, 10 μm. d A schematic diagram showing that pre-incubation with pre-rRNA blocks the binding of the recombinant BRCA1 BRCT or BARD1 BRCT to IRIF (top left panel). Foci were examined with anti-GST antibody (bottom panel). DSB foci were marked with an anti-γH2AX antibody. Foci number per cell is shown (top right panel). P values were calculated using Student’s t test. n.s. nonsignificant, * P < 0.05, *** P < 0.001. Scale bars, 10 μm. e Enrichment of pre-rRNA at the unsynapsed axis of X and Y chromosomes. Analysis of pre-rRNA and SCP3 distribution is shown (right panel). f Pre-incubation of the BRCTs with pre-rRNA abolishes their localization onto the XY body. Recombinant BRCT proteins were pre-incubated with pre-rRNA. The circled area indicates the XY body. Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Staining, Incubation, Binding Assay, Recombinant

Journal: Cell Discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: a The BRCT domains of BRCA1 or BARD1 form LLPS with pre-rRNA. Recombinant BRCA1 BRCT or BARD1 BRCT was incubated with pre-rRNA in the presence of a crowding agent (10% PEG 8000). LLPS was examined by microscopy after incubation on a coverslip for 10 min. Droplet formation and average droplet diameter were analyzed (right panels). Corresponding square networks consist of green and red dots representing droplet formation in indicated conditions. b LLPS of the BRCTs and pre-rRNA complex was abolished by RNase A treatment. Recombinant BRCA1 BRCT and BARD1 BRCT were incubated with pre-rRNA, followed by 1 mg/mL RNase A treatment. Statistical analysis of droplet formation is shown (right panel). Values are means ± SD of three independent assays. c Microscopy images of individual droplet fusions at indicated time points. d Microscopy images of individual droplet dynamic imaging at indicated time points followed by photobleaching. Statistical analysis of droplets fluorescence is shown (right panel). Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Recombinant, Incubation, Microscopy, Imaging, Fluorescence

Journal: Cell Discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: a The association between pre-rRNA and M1755R of the BRCA1 BRCT or C645R of the BARD1 BRCT was examined by protein pull-down assays. RT-qPCR was performed following the GST-protein pull-down assays. b The mutations of the BRCT domains drastically reduce their binding affinity with RNA. The binding affinities between the mutant BRCT domains and 25-nt biotin-labeled RNA oligos were measured by BLI assays. c The BRCT domain mutants fail to form LLPS droplets even in the presence of pre-rRNA. The droplet area was measured. Wild-type BRCTs forming droplets in the presence of 50 ng/µL pre-RNA was used as control. d The BRCT domain mutants fail to localize onto IRIF. Foci-positive cells were analyzed. Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Quantitative RT-PCR, Binding Assay, Mutagenesis, Labeling, Control

Journal: Advanced Functional Materials

Article Title: Biomimetic Nanoparticles as a Theranostic Tool for Traumatic Brain Injury

doi: 10.1002/adfm.202100722

Figure Lengend Snippet: Nanoparticles’ (NPs) properties characterization. After fabrication, NPs were characterized for their physicochemical and biological properties using dynamic light scattering. No significant changes in A) size and B) polydispersity index (PDI) were observed. However, a significant decrease in C) zeta potential for leukosomes (Leuko) compared to liposomes (Lipo) was noticed. D) Representative cryo‐TEM images of Lipo and Leuko verified that no morphological changes occurred following membrane protein integration to the NPs. Scale bars = 100 nm. E) Western blots for leukocyte membrane protein markers: CD11b, CD18, CD47, and CD45 indicated their membrane integration in Leuko but absence in Lipo. Results are shown as mean ± SEM. Unpaired t‐test was used to determine statistical probabilities * p ≤ 0.05 among means considered statistically significant, n = 5.

Article Snippet: Antibodies for western blot rat anti‐CD11b (MAB11241), goat anti‐CD18 (AF2618), rabbit anti‐CD45 (EPR20033), goat anti‐CD47 (ab108415), anti‐rabbit IgG‐HRP, anti‐goat IgG‐HRP, and anti‐rat IgG (Bio‐Techne Corporation, Minnesota, USA).

Techniques: Zeta Potential Analyzer, Liposomes, Membrane, Western Blot