Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Surface view of the 3D reconstruction. Two halves were colored yellow and cyan, representing two potential monomers although the exact boundary is unknown. ( b-g ) Antibody labeling against C-terminal Flag tag ( b-d ) and BRC repeats ( e-g ). In ( b ) and ( e ), raw particles with antibody are circled. ( c ) and ( f ) Reprojections along the same orientations of ( b) and ( e) . ( d ) and ( g ) 3D reconstructions viewed along the same directions as ( b) and ( e) , with antibody locations represented by spheres. ( h ) and ( i ) Top and side views of BRCA2 with antibody locations colored (Flag tag- blue, BRC – magenta). Magnification bars in all single particle images represent 100 Å.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Antibody Labeling, FLAG-tag, Single Particle

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) and ( b ) Side and top surface views of the 3D reconstruction. ( c ) Antibody labeling against RAD51. Left: individual particles with antibody circled. Middle: corresponding reprojections from the BRCA2-RAD51 reconstruction. Right: surface view along the same direction with antibody locations indicated with spheres. ( d ) Cylinders representing antibody locations defined from individual particles (upper). These intersect at the density regions on the outer rim connecting the two halves (lower, orange surface).

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Antibody Labeling

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Overlay of BRCA2 dimer (yellow and cyan) and BRCA2-RAD51 (pink mesh) highlighting the differences in their shape. ( b ) Rearranged BRCA2 dimer fitted into the BRCA2-RAD51 complex. ( c ) as in (b). Four RAD51 monomers (orange ribbon) were fitted into the additional density in BRCA2-RAD51 not accounted for by BRCA2 density. ( d ) Four RAD51 monomers, arranged as in filaments. (e) Histogram of mass measurement of BRCA2-RAD51 complex using STEM, showing peaks at 800 kD and 1200 kD, corresponding to BRCA2 dimer and BRA2 dimer binding to 8-10 RAD51.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Mass Measurement, Binding Assay

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Gel-shift assay showing the binding of BRCA2 to 5′- 32 P-labeled ssDNA substrates ranging from 20 to 100 nt. DNA was detected by autoradiography. ( b ) Images of individual particles of BRCA2 bound to gapped DNA (duplex arms are indicated in orange). Magnification bars represent 100 Å. ( c-e ) Electron microscopic visualization of RAD51-ssDNA filaments, BRCA2-ssDNA complexes, and BRCA2-RAD51-ssDNA complexes, as indicated. ( f ) Localization of BRCA2 in BRCA2-RAD51-ssDNA complexes by immunogold labeling. ( g ) Visualization of BRCA2-RAD51 filaments formed with 5′-gold particle labeled ssDNA. Magnification bars represent 100 nm.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Gel Shift, Binding Assay, Labeling, Autoradiography

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) and ( b ) Effect of BRCA2 on the number of RAD51-ssDNA nucleation events, as determined by electron microscopy. Inserts show enlargements of RAD51 filaments. ( c-d ) Quantification of RAD51-ssDNA filament length ( c ) and nucleation events ( d ) in the presence (blue) or absence (orange) of BRCA2, as determined by measurement of images shown in (n = 314) and 4 e (n = 332), n = number of RAD51 filaments. In total, 204 (RAD51-ssDNA) and 149 (BRCA2-RAD51-ssDNA) randomly collected grid areas were quantified. P-values (P < 0.0001) were determined using a two-tailed t test, error bars represent SD. ( e ) BRCA2-RAD51-ssDNA complexes visualized as multiple distinct filament nucleation sites on the same ssDNA molecule (arrowed). Magnification bars represent 100 nm.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Electron Microscopy, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Crystal structure of RPA bound to 30 nt ssDNA, showing a compact configuration and bending of the ssDNA into a U-shape. DNA binding domains of BRCA2 could adapt similar conformations. The polarity of the ssDNA is indicated. The two RPA molecules, related by 2-fold symmetry, could represent the DNA binding domains in the BRCA2 dimer as indicated below. ( b ) The DNA binding domains (1,2,3,4) of BRCA2 are depicted in similar conformations as those shown for RPA in (a), such that ssDNA could simultaneously bind to domains 3-4 (OB2-OB3) at the 5′ end (left hand side) of one BRCA2 monomer while domains 1-2 (alpha-helical domain and OB1) at the 3′ end (right hand side) of the second monomer. Two sets of RAD51 molecules bind the BRCA2 dimer in opposing directions. Only one set can be productive in ssDNA binding. ( c ) Model for filament formation and elongation using multiple BRCA2-RAD51 nucleation sites with BRCA2 acting as a molecular chaperone for RAD51.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Binding Assay

Journal: EMBO Reports

Article Title: Arginine methylation-dependent METTL14-SMN interaction regulates RNA m 6 A homeostasis

doi: 10.1038/s44319-025-00590-7

Figure Lengend Snippet: ( A ) Sanger sequencing confirmation of Mettl14 arginine methylation deficient mouse model. In total, 13 arginine resides located at the C-terminal disordered region of Mettl14 were mutated to lysine residues. ( B ) The arginine methylation levels of Mettl14 in WT, heterozygous (WT/RK), and homozygous (RK/RK) mice were detected by IP-western blot using tissue lysates from mouse spleen and thymus. ( C ) The protein expression levels of several DNA repair genes, including BRCA1, ATRIP, and PALB2, were detected by western blot using Thymus tissues from WT, WT/RK, and RK/RK mice. ( D ) Mettl14 arginine methylation deficiency increases the population of primitive hematopoietic cells (LK cells). ** P = 0.0019. ( E ) Mettl14 arginine methylation deficiency increases the population of myeloid (Mac1 + Gr1 + ) cells in the BM, but not in the spleen. * P = 0.013. ( F ) Mettl14 arginine methylation deficiency decreases the B lymphoid (B220 + ) population in the BM, but not in the spleen. * P = 0.011. ( G ) Mettl14 arginine methylation deficiency decreases the T lymphoid (CD3 + ) population in the BM, but not in the spleen. Data from three independent replicates were analyzed by Student’s t test and shown as mean ± SD. ** P = 0.0097. .

Article Snippet: Rabbit anti-PALB2 antibody , Proteintech , 14340-1-AP.

Techniques: Sequencing, Methylation, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: BRCA2 deficiency is a potential driver for human primary ovarian insufficiency

doi: 10.1038/s41419-019-1720-0

Figure Lengend Snippet: a The gene panel on the RT 2 Profiler PCR Array. These genes are crucial factors in BER, NER, MMR, DSB repair, or other relative pathways in DNA damage response. b Quality control test of the whole panel of the array. Five control genes (ACTB, B2M, GAPDH, HPRT1, and RPLPO) were contained on the array as quality control. Average CT value of the control genes were summarized from real-time qPCR of the array. All the included oocytes showed qualified CT value except oocyte POI-10 and oocyte POI-13. These two oocytes were thus excluded from the subsequent experiments. c Expression level of the 84 DNA damage response genes among the individual human oocytes. POI-04 and POI-20 oocytes showed significantly lower expression of BRCA2 , while POI-17 oocyte expressed lower XRCC2

Article Snippet: Rabbit polyclonal anti-Brca2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TA, USA; Cat#: sc-293185); mouse monoclonal anti-α-tubulin-fluorescein isothiocyanate (FITC) antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: F2168); Rabbit monoclonal anti-γH2AX and anti-Gapdh antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; Cat#: 9718, 2118); HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); FITC-conjugated goat anti-rabbit IgG (H + L), TRITC-conjugated goat anti-rabbit IgG (H + L) and FITC-conjugated goat anti-mouse IgG (H + L) were purchased from Zhongshan Golden Bridge Biotechnology Co., LTD (Beijing, China).

Techniques: Control, Expressing

Journal: Cell Death & Disease

Article Title: BRCA2 deficiency is a potential driver for human primary ovarian insufficiency

doi: 10.1038/s41419-019-1720-0

Figure Lengend Snippet: a Schematic illustration of deletion of Brca2 exons and creation of Brca2 knockout allele by Gdf9-Cre -mediated recombination in oocytes. b Immunoblotting analysis of Brca2 protein expression in control and Brca2 -deficient oocytes. The amount of Gapdh was used as an internal control. Molecular mass is given in kilodaltons. Two hundered germinal vesicle oocytes isolated from ovaries of 2-month-old mice were used for each lane of the blots. For each experiment, at least five mice of each genotype were used. c Comparison of breeding between control and Brca2 -deficient females. The female mice were cocaged with fertile male mice. The breeding assay was recorded for at least 6 months

Article Snippet: Rabbit polyclonal anti-Brca2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TA, USA; Cat#: sc-293185); mouse monoclonal anti-α-tubulin-fluorescein isothiocyanate (FITC) antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: F2168); Rabbit monoclonal anti-γH2AX and anti-Gapdh antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; Cat#: 9718, 2118); HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); FITC-conjugated goat anti-rabbit IgG (H + L), TRITC-conjugated goat anti-rabbit IgG (H + L) and FITC-conjugated goat anti-mouse IgG (H + L) were purchased from Zhongshan Golden Bridge Biotechnology Co., LTD (Beijing, China).

Techniques: Knock-Out, Western Blot, Expressing, Control, Isolation, Comparison

Journal: Cell Death & Disease

Article Title: BRCA2 deficiency is a potential driver for human primary ovarian insufficiency

doi: 10.1038/s41419-019-1720-0

Figure Lengend Snippet: a Representative images of ovarian size from control and Brca2 -deficient mice. Scale bar, 200 μm. b Average weights of ovaries obtained from control and Brca2 -deficient females. Data are shown as mean ± SEM. c – f Histology of ovarian sections from young adult control and Brca2 -deficient females. Ovaries from 2-month-old mice were embedded in paraffin, and sections of 8 μm thickness were prepared and stained with H&E. Black arrows in ( d ) show the growing follicles at different developmental stages; yellow arrows in ( f ) indicate the developmentally arrested follicles with degenerating oocytes. CL, corpus luteum. Scale bars in ( c ) and ( e ), 250 μm; scale bars in ( d ) and ( f ), 50 μm. g Quantification analysis of primordial follicles between control and Brca2 -deficient ovaries. h Quantification analysis of activated follicles between control and Brca2 -deficient ovaries. Brca2 -deficient ovary contained a significantly increased number of activated follicles with developmental arrest and degraded oocytes compared with the control ovary

Article Snippet: Rabbit polyclonal anti-Brca2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TA, USA; Cat#: sc-293185); mouse monoclonal anti-α-tubulin-fluorescein isothiocyanate (FITC) antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: F2168); Rabbit monoclonal anti-γH2AX and anti-Gapdh antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; Cat#: 9718, 2118); HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); FITC-conjugated goat anti-rabbit IgG (H + L), TRITC-conjugated goat anti-rabbit IgG (H + L) and FITC-conjugated goat anti-mouse IgG (H + L) were purchased from Zhongshan Golden Bridge Biotechnology Co., LTD (Beijing, China).

Techniques: Control, Staining

Journal: Cell Death & Disease

Article Title: BRCA2 deficiency is a potential driver for human primary ovarian insufficiency

doi: 10.1038/s41419-019-1720-0

Figure Lengend Snippet: a Representative images of in vivo matured eggs from control and Brca2 -deficient mice. Scale bar, 20μm. b The number of eggs obtained from control and Brca2 -deficient mice. c The rate of fragmented eggs retrieved from control and Brca2 -deficient mice. d Immunofluorescence of phosphorylated H2AX (γH2AX) in control and Brca2 -deficient oocytes. e Fluorescence intensity of γH2AX signal in control and Brca2 -deficient oocytes were summarized. f Representative images of GV oocytes obtained from control and Brca2 -deficient ovaries. Scale bar, 20 μm. g The rate of developmentally arrested oocytes in control and Brca2 -deficient mice. h Representative images of MII oocytes matured in vitro from control and Brca2 -deficient GV oocytes. Scale bar, 20 μm. i The rate of first polar body extrusion of control and Brca2 -deficient oocytes matured in vitro. j Representative images of spindle morphology and chromosome alignment in control and Brca2 -deficient oocytes after in vitro maturation. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles and counterstained with Hoechst to visualize chromosomes. Scale bar, 20 μm. k The percentage of oocytes with aberrant spindle in control and Brca2 -deficient groups. l The percentage of oocytes with misaligned chromosome in control and Brca2 -deficient groups. Statistical data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance

Article Snippet: Rabbit polyclonal anti-Brca2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TA, USA; Cat#: sc-293185); mouse monoclonal anti-α-tubulin-fluorescein isothiocyanate (FITC) antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: F2168); Rabbit monoclonal anti-γH2AX and anti-Gapdh antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; Cat#: 9718, 2118); HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); FITC-conjugated goat anti-rabbit IgG (H + L), TRITC-conjugated goat anti-rabbit IgG (H + L) and FITC-conjugated goat anti-mouse IgG (H + L) were purchased from Zhongshan Golden Bridge Biotechnology Co., LTD (Beijing, China).

Techniques: In Vivo, Control, Immunofluorescence, Fluorescence, In Vitro

Journal: Cell Death & Disease

Article Title: BRCA2 deficiency is a potential driver for human primary ovarian insufficiency

doi: 10.1038/s41419-019-1720-0

Figure Lengend Snippet: a Immunoblotting analysis of Brca2-HA protein expression in Brca2 -deficient oocytes. The blot was probed with anti-Brca2 antibody. The amount of Gapdh was used as an internal control. Two hundered germinal vesicle oocytes isolated from ovaries of 2-month-old mice were used for each lane of the blots. For each experiment, at least five mice of each genotype were used. b Representative images of MII oocytes matured in vitro from control ( Brca2 -proficient), Brca2 -deficient, and Brca2-rescued GV oocytes. Scale bar, 20 μm. c The rate of first polar body extrusion of control, Brca2 -deficient, and Brca2-rescued oocytes matured in vitro. d Representative images of spindle morphology and chromosome alignment in control, Brca2 -deficient, and Brca2-rescued oocytes after in vitro maturation. Oocytes were immunostained with anti-α-tubulin-FITC antibody to visualize spindles and counterstained with Hoechst to visualize chromosomes. Scale bar, 20 μm. ( e , f ) The rates of disorganized spindles and misaligned chromosomes in control, Brca2 -deficient and Brca2-rescued oocytes. g Immunofluorescence of γH2AX in control, Brca2 -deficient and Brca2-rescued oocytes. h Fluorescence intensity of γH2AX signal in control, Brca2 -deficient and Brca2-rescued oocytes were summarized. Statistical data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a p < 0.05 level of significance

Article Snippet: Rabbit polyclonal anti-Brca2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TA, USA; Cat#: sc-293185); mouse monoclonal anti-α-tubulin-fluorescein isothiocyanate (FITC) antibody was purchased from Sigma (St. Louis, MO, USA; Cat#: F2168); Rabbit monoclonal anti-γH2AX and anti-Gapdh antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA; Cat#: 9718, 2118); HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); FITC-conjugated goat anti-rabbit IgG (H + L), TRITC-conjugated goat anti-rabbit IgG (H + L) and FITC-conjugated goat anti-mouse IgG (H + L) were purchased from Zhongshan Golden Bridge Biotechnology Co., LTD (Beijing, China).

Techniques: Western Blot, Expressing, Control, Isolation, In Vitro, Immunofluorescence, Fluorescence

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 1. Depletion of RFWD3 rescues HU sensitivity, nascent DNA degradation, and stalled fork collapse in BRCA2-deficient cells. (A) Detection of RFWD3 and BRCA2 levels in U2OS cells used in Fig. 1 B. (B) HU sensitivity of U2OS cells transfected with siBRCA2-3 and /or siRFWD3-4. Cell survival is normalized to the untreated control for each siRNA condition. Data represent the mean and SD of three replicates per HU dose and siRNA condition. Asterisks indicate P-values for RFWD3/BRCA2 versus BRCA2 depletion using an unpaired t test (*P < 0.05; ***P < 0.001; ****P < 0.0001). Data are representative of three independent experiments, for which mean LC50 values are provided in Fig. S1 B. (C) Schematic for single DNA fiber analysis to detect nascent DNA degradation at stalled forks. U2OS cells transfected with siBRCA2-3 and /or siRFWD3-4 were labeled with sequential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h). Representative images are provided for replication tracks containing both CldU and IdU from cells transfected with the indicated siRNAs. Scale bars, 5 µm. (D) IdU/CldU replication track ratios in U2OS cells treated as in Fig. 1 C. Median values from >200 replication tracks are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (E) Representative images of neutral comet tails in U2OS cells transfected with siBRCA2- 3 and/or siRFWD3-4 and treated with HU for 24 h. Scale bars, 50 µm. (F) Box plot of neutral comet-tail moments in U2OS cells from Fig. 1 E. Whiskers represent the 10th and 90th percentiles. More than 300 cells were scored for each condition (n.s., not significant; ****P < 0.0001; Mann Whitney test).

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Transfection, Control, Labeling, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 2. Mutations in the RFWD3 ubiquitin ligase and WD40 domains rescue nascent DNA degradation in BRCA2-deficient cells. (A) Detection of RFWD3 and BRCA2 levels in U2OS cells for the experiment in Fig. 2 B. (B) U2OS cells expressing siRNA-resistant RFWD3 (WT or C315A) were transfected with siBRCA2-3 and siRFWD3-4, and they were compared with U2OS cells transfected with siBRCA2-3 with or without siRFWD3-4. Cells were labeled with se- quential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h) as in Fig. 1 C. Median values for IdU/CldU track ratios (from >200 tracks) are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (C) Detection of RFWD3 and BRCA2 levels in FA patient fibroblasts for the experiment in Fig. 2 D. (D) Schematic for single DNA fiber analysis to detect nascent DNA degradation at stalled forks in FA patient fibroblasts complemented with WT RFWD3 (1143 + WT) or empty vector (1143 + mock). Cells were labeled with sequential CldU (40 min) and IdU (50 min) and then treated with 2 mM HU (5 h). Representative images are provided for CldU and IdU-containing replication tracks upon transfection with siFF or siBRCA2-3. Scale bars, 5 µm. (E) IdU/ CldU replication length ratios in FA patient fibroblasts treated as in Fig. 2 D. Median values from >200 replication tracks are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test).

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Labeling, MANN-WHITNEY, Plasmid Preparation

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 6. RFWD3 stimulates ZRANB3 recruitment and replication fork reversal in BRCA2-deficient cells. (A) Immunoblot showing RFWD3, BRCA2, and HA-ZRANB3 levels in U2OS cells used in Fig. 6, B–E. HA-ZRANB3 was detected with anti-HA antibody. (B) U2OS cells expressing HA-ZRANB3 were transfected with siRFWD3-2 and/or siBRCA2-3, fixed 30 min after UV laser irradiation, and stained with anti-HA (green) and anti-γH2AX (red) antibodies. Scale bars, 10 µm. (C) Graph showing the percentage of γH2AX-positive cells with HA-ZRANB3 colocalization at UV laser stripes corresponding to the experiment in Fig. 6 B. Data represent the mean and SD from two independent experiments (**P < 0.01, unpaired t test). (D) U2OS cells expressing HA-ZRANB3 were transfected with siRFWD3-2 and/or siBRCA2-3 and treated with 10 µM EdU for 10 min followed by 1 µg/ml 4NQO for 4 h. After cell fixation, biotin was conjugated to EdU by click chemistry, and proximity ligation assay (PLA) was performed with anti-HA and anti-biotin antibodies. Images are representative of results quantitated in Fig. 6 E. Scale bars, 5 µm. (E) Box plot showing the distribution of PLA foci per cell for each condition (>200 cells) in Fig. 6 D. Whiskers represent the 10th and

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Western Blot, Expressing, Transfection, Irradiation, Staining, Proximity Ligation Assay

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 7. RFWD3 and ZRANB3 epistasis in replication fork remodeling phenotypes. (A) Detection of RFWD3, ZRANB3, and BRCA2 levels in U2OS cells used in Fig. 7, B and C. (B) U2OS cells were transfected with siBRCA2-3, siRFWD3-4, and/or siZRANB3. They were labeled with sequential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h) as in Fig. 1 C. Median values for IdU/CldU track ratios (from >200 tracks) are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (C) Neutral comet-tail moments in U2OS cells transfected with siBRCA2-3, siRFWD3-4, and/or siZRANB3 and treated with 2 mM HU for 24 h. Whiskers represent the 10th and 90th percentiles. More than 200 cells were analyzed for each condition (****P < 0.0001, Mann Whitney test). (D) Detection of RFWD3 and ZRANB3 levels in U2OS cells used in Fig. 7 E. (E) U2OS cells transfected with siRFWD3-4 and/or siZRANB3 were treated with 2 mM HU for 5 h. Replication intermediates were detected by electron microscopy, and the percentage of reversed forks was measured in a single replicate. The number of replication intermediates analyzed for each condition is indicated in parentheses.

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Transfection, Labeling, MANN-WHITNEY, Electron Microscopy

Journal: Gynecologic Oncology Reports

Article Title: BRCA immunohistochemistry for screening of BRCA mutation in epithelial ovarian cancer patients

doi: 10.1016/j.gore.2020.100582

Figure Lengend Snippet: Performance of BRCA1 immunohistochemistry (IHC) and BRCA mutation status.

Article Snippet: Samples were stained and incubated for 60 min with BRCA1 mouse monoclonal antibody (Novus biological Inc., USA) and BRCA2 rabbit polyclonal antibody (Novus biological Inc., USA) at a dilution ratio of 1:100.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Gynecologic Oncology Reports

Article Title: BRCA immunohistochemistry for screening of BRCA mutation in epithelial ovarian cancer patients

doi: 10.1016/j.gore.2020.100582

Figure Lengend Snippet: Performance of BRCA2 immunohistochemistry (IHC) and BRCA mutation status.

Article Snippet: Samples were stained and incubated for 60 min with BRCA1 mouse monoclonal antibody (Novus biological Inc., USA) and BRCA2 rabbit polyclonal antibody (Novus biological Inc., USA) at a dilution ratio of 1:100.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Gynecologic Oncology Reports

Article Title: BRCA immunohistochemistry for screening of BRCA mutation in epithelial ovarian cancer patients

doi: 10.1016/j.gore.2020.100582

Figure Lengend Snippet: Performance of BRCA 1/2 immunohistochemistry (IHC) for detection of germline BRCA1/2 mutation.

Article Snippet: Samples were stained and incubated for 60 min with BRCA1 mouse monoclonal antibody (Novus biological Inc., USA) and BRCA2 rabbit polyclonal antibody (Novus biological Inc., USA) at a dilution ratio of 1:100.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Journal of International Society of Preventive & Community Dentistry

Article Title: BRCA1/2 Mutations in Salivary Pleomorphic Adenoma and Carcinoma-ex-Pleomorphic Adenoma

doi: 10.4103/jispcd.JISPCD_184_17

Figure Lengend Snippet: Higher magnification indicates BRCA2 nuclear positivity in myoepithelial cells and the cells in the chondroid material in pleomorphic adenoma samples

Article Snippet: Slides were incubated with primary mouse monoclonal anti-BRCA1 antibody (Abcam, ab16780) at 1:90 dilution and primary rabbit polyclonal anti-BRCA2 antibody (Abcam, ab 27976) at 1:80 for 1 h at room temperature, followed by incubation with secondary antibody for 30 min and immunostained with DAB (3, 3’-diaminobenzidine) for 5 min as a chromogen and hematoxylin as counterstain.

Techniques:

Journal: Journal of International Society of Preventive & Community Dentistry

Article Title: BRCA1/2 Mutations in Salivary Pleomorphic Adenoma and Carcinoma-ex-Pleomorphic Adenoma

doi: 10.4103/jispcd.JISPCD_184_17

Figure Lengend Snippet: A lower magnification indicates a strong BRCA2 positivity in polymorphous low-grade adenocarcinoma subtype. Notice the single-file cells and infiltrative invasive front

Article Snippet: Slides were incubated with primary mouse monoclonal anti-BRCA1 antibody (Abcam, ab16780) at 1:90 dilution and primary rabbit polyclonal anti-BRCA2 antibody (Abcam, ab 27976) at 1:80 for 1 h at room temperature, followed by incubation with secondary antibody for 30 min and immunostained with DAB (3, 3’-diaminobenzidine) for 5 min as a chromogen and hematoxylin as counterstain.

Techniques: