Journal: Cell Death & Disease

Article Title: Di (2-ethylhexyl) phthalate exposure impairs meiotic progression and DNA damage repair in fetal mouse oocytes in vitro

doi: 10.1038/cddis.2017.350

Figure Lengend Snippet: Exposure to DEHP impairs DNA repair in oocytes. ( a ) Immunolabeling of the oocyte chromosomes with anti-SYCP3 (red) and anti-BRCA1 (green) antibodies. ( b ) Percentage of BRCA1 positive oocytes after six days of culture with or without DEHP ( c ) Percentage of oocytes at the pachytene and diplotene stages showing negative or positive BRCA1 staining after 6 days of culture with or without DEHP. (* P <0.05; ** P <0.01)

Article Snippet: Blocking was performed by dipping the slides in antibody dilution buffer (ADB; 0.3% BSA, 10% normal goat serum and 0.005% Triton-X-100 in TBS) for 30 min before incubation in primary antibody, anti-SCP3 at a dilution of 1:150 (Abcam, ab97672, San Francisco, CA, USA; or Novus, NB300-232, Littleton, CO, USA), anti- γ H2AX at a dilution of 1:150 (Abcam, ab26350), anti-RAD51 at a dilution of 1:150 (Abcam, ab133534), BRCA1 at a dilution of 1:100 (Boster, PB9015, Wuhan, China), or anti-MLH1 at a dilution of 1:150 (BD Pharmingen, 551091, Franklin Lakes, NJ, USA) for 3 h at 37 °C.

Techniques: Immunolabeling, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Steady‐state levels of gastric cancer cell lines. Using Western blot assay, steady protein levels of BRCA1, BRCA2 and c‐MET are analysed in primary gastric cancer cell lines HS746T and AGS. Protein levels were normalized against actin

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Low levels of c‐MET partially sensitize GC cell lines in PARP inhibition. A, HS746T/AGS cells, control‐siRNA‐Hs746T/AGS cells and si‐c‐MET Hs746T/AGS cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of c‐MET expression (by Western blot analysis) revealed down‐regulation of the c‐MET receptor in both cell lines (HS746T and AGS); (B) HS746T cells, control‐siRNA‐Hs746T cells and si‐BRCA1/2 Hs746T cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of BRCA1 and BRCA2 expression (by Western blot analysis) revealed down‐regulation of the BRCA1/2 in HS746T cell line; (C) HS746T cells, control‐siRNA‐Hs746T, siBRCA1/2‐Hs746T and siMET/BRCA1/2‐Hs746T cells were cultured with the indicated concentrations of NU1025 (5, 10 and 20 μmol/L) for 48 h for determination of cell viability (MTT metabolic activity assay). Error bars represent SD

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Inhibition, Control, Metabolic Assay, Expressing, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

doi: 10.1111/jcmm.15655

Figure Lengend Snippet: Co‐inhibition of c‐MET (SU11274) and PARP (NU1025) sensitizes GC cells after knockdown BRCA1/2. Knocking down BRCA1 or BRCA2 sensitizes cells to PARP and c‐MET inhibition in HS746T cells expressing low levels of c‐MET (AGS cells, c‐MET knockdown Hs746T cells) to PARP inhibition. A, HS746T cells, control‐siRNA‐Hs746T cells and siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cells were exposed to 5 µmol/L of NU1025 and/or 5 µmol/L of SU11274 for 48 h for determination of cell viability (MTT metabolic activity assay). Results are expressed as percentages. Average values of three experiments ± SD are shown; (B) Western blot analysis of PARP and cl.caspase‐3 in Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cell lines. Cells were cultured with the indicated drugs (5 μmol/L NU1025, 5 μmol/L SU11274 alone or in combination for 24 h of treatment). Protein levels were normalized against actin

Article Snippet: Antibodies were used against: BRCA1 (#MCA5946GT) was purchased from Bio‐Rad, BRCA2 (sc‐295185), actin (sc‐8035) and histone H2AX (#sc‐517336) were purchased from Santa Cruz Biotechnology, and PARP‐1 (#9542), cleaved caspase‐3 (#9661) and c‐Met (#8198) from Cell Signaling Technology.

Techniques: Inhibition, Knockdown, Expressing, Control, Metabolic Assay, Western Blot, Cell Culture

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Cross-analysis of the methylation of RAD51 and BRCA1 genes in tumor and non-tumor mucosa, considering the 37 cases with paired tumoral and non-tumoral samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Methylation

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in gastric adenocarcinoma and non-tumor gastric mucosa samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison of RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in tumor and non-tumor mucosa, considering the paired samples of the same individuals. The results are presented in box plots, using a logarithmic scale (+1 was added as a constant to allow the logarithmic representation of null values).

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression in patients with previous neoadjuvant chemotherapy and in those without neoadjuvant chemotherapy, considering tumor samples and non-tumor mucosa samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Association between immunohistochemistry and mRNA levels in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Immunohistochemistry

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Correlation between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression and histological type, histological grade, perineural invasion, and vascular invasion in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Correlation between RAD51 , ATR , BRCA1 , BRCA2 , and ATM mRNA expression and depth of invasion (pT), regional lymph node metastasis, distant metastasis, and stage in tumor samples.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Comparison between the mRNA levels in the tumors of patients who died and those who did not, considering the following groups: total population, excluding neoadjuvant therapy, with only surgical treatment, and with adjuvant or neoadjuvant treatment.

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Comparison, Adjuvant

Journal: Biomarker Insights

Article Title: mRNA Expression and Methylation of the RAD51 , ATM , ATR , BRCA1 , and BRCA2 Genes in Gastric Adenocarcinoma

doi: 10.1177/11772719231225206

Figure Lengend Snippet: Kaplan-Meier curves comparing the overall survival of the 24 patients with tumor mRNA expression above the median with that of the 24 below the median, for RAD51 , ATR , BRCA1 , BRCA2 , and ATM .

Article Snippet: The following antibodies were used: ATM (St John’s Laboratory, London, UK, STJ97797, 1:400), ATR (Abcam, Cambridge, UK, ab178407, 1:100), BRCA1 (St John’s Laboratory, STJ113833, 1:300), RAD51 (St John’s Laboratory, STJ95330, 1:100), and BRCA2 (St John’s Laboratory, STJ91885, 1:100).

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells.

doi: 10.1095/biolreprod.107.063107

Figure Lengend Snippet: FIG. 1. Purification and functional char- acterization of dimeric TRF. A) Chromato- graphic profile of highly purified human TRF. The TRF solution was loaded onto an ultragel Aca44 column as described in Materials and Methods. OD, optical density. B) Each fraction was analyzed using native gel electrophoresis and immunoblotting. VLMB, very low-mobility band; LMB, low- mobility band; HMB, high-mobility band. C) Sensorgrams of interaction between high- and low-mobility purified fractions of hTRF and the monoclonal anti-hTRF anti- body. The interaction between both forms of hTRF and the monoclonal antibody is indicated by a dashed line. After reinjection of the monoclonal antibody, only the accessible epitope on the dimeric form generated a secondary signal (dark line), in contrast to the monomeric form (light line). The arrows directed to the top indicate the beginning of injection of the anti-hTRF antibody, and those directed toward the bottom indicate the end of the injections. The principle of discrimination between monomeric versus dimeric TRF by SPR is schematized on the right. RU, resonance units.

Article Snippet: Monoclonal mouse antibodies raised against the N-terminal domain of human TRF were from AbD Serotec (Cergy Saint-Christophe, France).

Techniques: Purification, Functional Assay, Nucleic Acid Electrophoresis, Western Blot, Generated, Injection

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A (young group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). B (young group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the nucleus (× 400). C (old group) BRCA1 protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). D (young group) WWOX protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400). E (old group) WWOX protein immunohistochemical staining was positive. The positive products localized in the cytoplasm (× 400) . F (young group) ER protein immunohistochemical staining was positive. The positive product positioning in the nucleus (× 400). G (young group) positive immunohistochemical staining of PR proteins. The positive products localized in the nucleus (× 400) H (young group) C-erbB2 protein immunohistochemical staining was positive. The positive products localized in the membrane (× 400). I (young group) Ki67 protein immunohistochemical staining was positive. The positive products localized in the nucleus (× 400).

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: Analysis of the correlation between the BRCA1 results and clinicopathological parameters in the young groups and old groups

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques:

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A The PCR product of exon 2 of the BRCA1 gene (259 bp). B The PCR product of exon 20 of the BRCA1 gene (401 bp).

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques:

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: A The partial DNA sequencing results of exon 2 of BRCA1 gene of patient 6. B The partial DNA sequencing results of exon 20 of BRCA1 gene of patient 6.

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: DNA Sequencing

Journal: Diagnostic Pathology

Article Title: The ectopic expression of BRCA1 is associated with genesis, progression, and prognosis of breast cancer in young patients

doi: 10.1186/1746-1596-7-181

Figure Lengend Snippet: Comparative analysis of the immunohistochemical results of young and old patient groups

Article Snippet: The WWOX antibody was purchased from Beijing Boaosen Biotechnology Co., Ltd. BRCA1 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. ER, PR, and Ki67 were expressed in the nucleus.

Techniques: Immunohistochemical staining

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A-D) Cytoplasmic and nuclear fractions from uninfected BJAB, HMVEC-d and HFF cells were immunoprecipitated (IP-ed) using anti-IFI16 and anti-H2B antibodies and immunoblotted for IFI16, H2B, BRCA1, H2A and ASC. The nuclear fraction from HMVEC-d cells was IP-ed with control IgG antibody for specificity control. (E and F) Uninfected cytoplasmic and nuclear fractions were western blotted with the above antibodies for equal inputs. TBP and Tubulin were used to monitor the purity of the nuclear and cytoplasmic fractions, respectively. (G and H) Uninfected BJAB, HMVEC-d and HFF cells were subjected to PLA reactions using anti-IFI16 and H2B antibodies as described in Materials and Methods. Boxed areas were enlarged and interactions of IFI16 with H2B are indicated by the red arrows. Bar diagrams represent quantitation of the average number of dots per cell in the cytoplasm and nucleus of uninfected cells. (I and J) The above cells were also tested by PLA using anti-IFI16, BRCA1 and H2B antibodies. Boxed areas were enlarged and localization of IFI16 with BRCA1 and H2B with BRCA1 are indicated by the red arrows. Nuclei were stained with DAPI.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Immunoprecipitation, Control, Western Blot, Quantitation Assay, Staining

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A-D) Nuclear and cytoplasmic fractions from uninfected and KSHV (30 DNA copies/cell) infected HMVEC-d cells at various time points (2, 4, 12, 24 h p.i.) were IP-ed using anti-IFI16 and anti-H2B antibodies and immunoblotted for IFI16, H2B, BRCA1, H2A and ASC. (E and F) Nuclear and cytoplasmic fractions were analyzed by WB for input controls, while tubulin and TBP WB showed the purity of cytoplasmic and nuclear fractions, respectively. (G and H) PLA analysis for the interaction of IFI16 with H2B at various time points of KSHV post-infection in HMVEC-d cells. Uninfected and KSHV (30 DNA copies/cell) infected for 4 h (G) and for 2, 12 and 24 h p.i. (H) HMVEC-d cells were subjected to PLA reactions using anti-IFI16 (mouse) and anti-H2B (rabbit) antibodies. Boxed areas were enlarged and the nuclear and cytoplasmic localization of IFI16-H2B are indicated by red and yellow arrows, respectively. (I-K) PLA analysis for the association and redistribution of H2B-IFI16 during HSV-1 de novo infection. Uninfected and HSV-1 infected (1 pfu/cell) (4 h) HFF cells were subjected to PLA reactions using anti-H2B, anti-IFI16, and anti-BRCA1 antibodies. Boxed areas were enlarged and H2B-IFI16, H2B-BRCA1 and IFI16-BRCA1 localization in the nucleus and cytoplasm are indicated by red and yellow arrows, respectively. Nuclei were stained with DAPI. Magnification: 40X.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Infection, Staining

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A and B) Cytoplasmic and nuclear extracts from BJAB and BCBL-1 cells were IP-ed using anti-IFI16 and anti-H2B antibodies, and immunoblotted with anti-IFI16, H2B, BRCA1, H2A and ASC antibodies. (C) Cytoplasmic and nuclear fractions were analyzed by WB for equal inputs, while tubulin and TBP confirmed the purity of cytoplasmic and nuclear extracts, respectively. (D-G) BJAB and BCBL-1 cells were tested by PLA using anti-IFI16 (mouse), anti-H2B (rabbit) and anti-H2A (rabbit) antibodies. Nuclear and cytoplasmic localization of IFI16-H2B and IFI16-H2A are indicated by white and red arrows, respectively (D and F). Nuclei were stained with DAPI. The average number of dots per cell in the nucleus and cytoplasm was quantitated and represented by bar graph (E and G). *, p<0.05; **, p<0.01; ***, p<0.001 nuclear vs. cytoplasmic dots.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Staining

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A, B and C) Cytoplasmic fractions from uninfected and KSHV infected HMVEC-d cells at 2, 4, 12, and 24 h p.i. were taken from and IP-ed using anti-IFI16, H2B and STING antibodies and western blotted for IFI16, H2B, and STING. (D) The cytoplasmic fraction was immunoblotted for STING for input control and tubulin and TBP for purity of the fraction. (E and F) Uninfected and KSHV infected (4 h) HMVEC-d were tested by PLA using anti-IFI16, H2B and STING antibodies. Boxed areas were enlarged. Red arrows represent the localization of IFI16-STING and H2B-STING in the cytoplasm. (G and H) PLA analysis demonstrating the association of IFI16-STING and H2B-STING during HSV-1 de novo infection. Uninfected and HSV-1 infected (1 pfu/cell) (4 h) HFF cells were subjected to PLA reactions. Boxed areas were enlarged and localization of IFI16-STING and H2B-STING in the cytoplasm is represented by red arrows. Magnification: 40X. (I, J and K) IFI16 and H2B association with STING during KSHV latent infection. Cytoplasmic and nuclear extracts from BJAB and BCBL-1 cells shown under experiments were IP-ed using anti-IFI16, H2B and STING antibodies and western blotted for IFI16, H2B, and STING. (L) Equal input of STING analyzed by WB. (M and N) BJAB and BCBL-1 cells were tested by PLA reactions using anti-IFI16 (mouse), H2B (goat) and STING (rabbit) antibodies. Red arrows represent the localization of IFI16-STING and H2B-STING in the cytoplasm. Nuclei were stained with DAPI. Magnification: 40X. (O) Association of cGAS with IFI16, H2B, STING and BRCA1 during KSHV de novo infection. Cytoplasmic fractions from uninfected and KSHV infected HMVEC-d cells at 2, 4, 12, and 24 h p.i. were IP-ed using anti-cGAS antibody and western blotted for IFI16, H2B, STING, BRCA1 and cGAS. (P) Input control for cGAS in the cytoplasmic fraction by WB. (Q and R) Association of cGAS with IFI16, H2B, STING and BRCA1 during KSHV latent infection. (Q) Cytoplasmic and nuclear fractions from BJAB and BCBL-1 cells were IP-ed with anti-cGAS antibody and western blotted for IFI16, H2B, STING, BRCA1 and cGAS. (R) Western blot showing input control for cGAS in cytoplasmic and nuclear fractions.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Infection, Western Blot, Control, Staining

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A) For a double PLA reaction, two independent reactions were performed. In the first PLA reactions, uninfected and KSHV (KS) infected (4 h) HMVEC-d cells were immunostained using mouse (ms) anti-IFI16 and goat (g) anti-H2B antibodies, and detected by DUOLink green detection agent. Cells were washed, blocked and subjected to the second PLA reactions using goat anti-H2B and rabbit (rb) anti-STING antibodies and visualized with red detection agents. Green and red dots indicate the localization of IFI16-H2B and H2B-STING, respectively. (B-E) Similarly, double PLA for different combinations were performed using BRCA1 (ms) + H2B (g) and H2B (g) + STING (rb); IFI16 (ms) + H2B (rb) and H2B (rb) + cGAS (g); BRCA1 (ms) + H2B (rb) and BRCA1 (ms) + cGAS (g); and H2B (ms) + STING (rb) and STING (rb) + cGAS (g). Red arrows indicate yellow dots representing the associations of three different proteins together such as IFI16-H2B-STING (A), BRCA1-H2B-STING (B), IFI16-H2B-cGAS (C), BRCA1-H2B-cGAS (D) and H2B-STING-cGAS (E) in the cytoplasm of KSHV infected cells. Nuclei were stained with DAPI. (F and G) Effect of BRCA1, cGAS and H2B on the interaction of IFI16-STING during KSHV de novo infection. (F) WCL from HMVEC-d cells electroporated with siC, siBRCA1, sicGAS and siH2B for 48 h then infected or uninfected with KSHV for 4 h were western blotted with anti-BRCA1, cGAS and H2B antibodies. (G) The WCL lysates from panel F were IP-ed with anti-IFI16 antibody and immunoblotted for STING and H2B. The bottom panels showed H2B knockdown efficiency and STING as an input control. (H and I) Role of ASC in the association of STING with IFI16 and H2B during KSHV de novo infection. WCL from HMVEC-d cells electroporated with siC or siASC for 48 h followed by with and without KSHV infections (4 and 24 h) were IP-ed with anti-STING antibody and immunoblotted for IFI16, H2B and STING. The bottom panels show the knockdown efficiency of ASC along with equal inputs of IFI16, H2B and STING by WB.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Infection, Staining, Western Blot, Knockdown, Control

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A) WCL from HMVEC-d cells electroporated with siC, siH2B, siIFI16, siSTING or siASC for 48 h followed by infection for 4 h with or without KSHV (KS) were western blotted with anti-IFI16, ASC, H2B and STING antibodies. (B) Phosphorylation of TBK1 and IRF3 in H2B, IFI16, BRCA1, cGAS, STING and ASC knockdown cells infected with KSHV. HMVEC-d cells electroporated with different siRNA (panel A and ) for 48 h were followed by with or without KSHV infection (4 h), and immunoblotted using anti-pTBK1, tTBK1 (total), pIRF3 and tIRF3 antibodies. (C and D) Effect of IFI16, H2B, BRCA1, cGAS, STING and ASC knockdown on IFN-β and IL-1β secretion during KSHV de novo infection. (C) Cell culture supernatants from HMVEC-d cells electroporated with siC, siIFI16, siBRCA1, sicGAS, siSTING, siH2B or siASC for 48 h followed by infection (4 h) with or without KSHV (Fig 7A and ), were collected and subjected to IFN-β ELISA. Results presented are means ± SD (* p<0.05, ** p<0.01 from siC vs. siIFI16, siBRCA1, sicGAS, siSTING, and siH2B with KSHV infection; NS: not significant). (D) The same supernatants from panel C were used to detect IL-1β secretion by ELISA. Results presented are means ± SD (* p<0.05, from siC vs. siIFI16, and siASC with KSHV infection; NS: not significant). (E) Effect of IFI16, H2B, BRCA1, cGAS, STING and ASC knockdown on IFN-β secretion during HSV-1 infection. Cell culture supernatants from HFF cells electroporated with siRNA for 48 h followed by with or without HSV-1 infection (4 h) were collected and IFN-β ELISA was performed. Results presented are means ± SD (*** p<0.001 from siC vs. siIFI16, siBRCA1, sicGAS, siSTING, or siH2B with HSV-1 infection; NS: not significant).

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Infection, Western Blot, Phospho-proteomics, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A) HMVEC-d cells were infected by EdU labeled or unlabeled KSHV genome (200 DNA copies/cell) for 2 h and (B) HFF cells were infected by EdU labeled or unlabeled HSV-1 genome (10 PFU/cell) for 2 h. Protein-DNA cross-linking was performed, and biotin-TEG azide selectively linked to the reactive alkyne group of EdU containing DNA through a click reaction. DNA was sheared and short chromatin fragments captured on streptavidin beads. Pulled down proteins were analyzed by immunoblot using anti-H2B and H3 antibodies. (C-F) H2B is critical in KSHV or HSV-1 genome recognition by IFI16 during de novo infection. (C) HMVEC-d cells electroporated with siC, siH2B or siBRCA1 for 48 h were infected by EdU-labeled KSHV (30 DNA copies/cell) for 2 h and tested by PLA using mouse and rabbit anti-IFI16 (green dots). EdU labeled KSHV genome was detected by reaction with Alexa 594 labeled picolylazide (red dots). The presence of IFI16 and EdU labeled KSHV genome association (yellow color) was indicated by white arrows. Red arrows in the left panel indicate the distribution of IFI16 in the cytoplasm. (D) Bar graph showing the quantitation of IFI16-EdU-KSHV genome colocalization (yellow dots). ** p<0.01 and *** p<0.001 that represents siC vs. siH2B and siBRCA1 with EdU-KSHV infection. (E) HFF cells electroporated using siC, siH2B or siBRCA1 for 48 h were infected by EdU-labeled HSV-1 (KOS) with 1 PFU/cell for 2 h and tested for PLA as described in panel (C). The presence of IFI16 and EdU labeled HSV-1 genome association (yellow dots) is indicated by white arrows. Nuclei were stained with DAPI. (F) Average colocalized yellow dots per cell were quantitated and presented by bar graph. ** p<0.01 and *** p<0.001 that presents siC vs. siH2B and siBRCA1 with EdU-HSV-1 infection. (G) HFF cells electroporated with siC, siBRCA1 or siH2B for 48 h were infected by EdU-labeled or unlabeled HSV-1 genome (10 PFU/cell) for 2 h. Protein-DNA cross-linking was performed as described in panel B. Pulled down protein was analyzed by immunoblot using anti-H2B and anti-IFI16 antibodies. The bottom panel shows the input controls for IFI16, H2B, BRCA1 and H3. (H) Effect of BRCA1 on KSHV genome recognition by IFI16-H2B during de novo infection. HMVEC-d cells electroporated with siC or siBRCA1 for 48 h were infected by EdU-labeled KSHV (30 DNA copies/cell) for 2 h and tested by PLA using mouse anti-IFI16 and rabbit anti-H2B (green dots). EdU labeled KSHV genome was detected by red dots. Boxed areas were enlarged and the presence of IFI16-H2B and EdU labeled KSHV genome association (yellow color) was indicated by white arrows (bottom panel). Red arrows in the top panel indicate the redistribution of IFI16-H2B in the cytoplasm. (I) The average number of colocalized yellow PLA dots per cell as shown in Fig 9H was quantitated and presented in the bar graphs. **p<0.01 from siC vs. siBRCA1 with EdU KSHV infection.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Infection, Labeling, Western Blot, Quantitation Assay, Staining

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: (A) HMVEC-d cells electroporated with siC or siBRCA1 for 48 h were uninfected or infected by KSHV for 4 h and subjected to PLA using mouse anti-IFI16 and rabbit anti-acetyl lysine antibodies. The localization of IFI16-acetyl lysine (acetylation of IFI16) PLA spots are shown in red. (B) WCL from HMVEC-d cells electroporated with siC and siBRCA1 for 48 h followed by with or without KSHV infection for 30’ (30 min), 4 and 24 h were IP-ed using anti-IFI16 and anti-acetyl lysine antibodies and western blotted for IFI16, BRCA1, p300, and H2B. Bottom input control panels show the levels of BRCA1, IFI16, p300 and total protein acetylation. (C) Schematic model illustrating the essential role of histone H2B in IFI16-mediated viral DNA genome sensing and innate IFN-β production during KSHV or HSV-1 de novo infection. Soon after KSHV or HSV-1 DNA entry into the nucleus, IFI16 in complex with BRCA1-H2B or with BRCA1 recognizes the viral genome, leading into BRCA1 mediated p300 recruitment, interaction with IFI16, acetylation (Ac) of IFI16 and H2B by p300, and cytoplasmic transport of acetylated IFI16-H2B-BRCA1 via Ran GTP. C646 or LPT treatments abolish acetylation or cytoplasmic translocation of H2B and IFI16, respectively. The inflammasome (IFI16-BRCA1-ASC-procaspase-1) independent IFI16-H2B-BRCA1 complex in the cytoplasm associates with cGAS and STING to form a macromolecular complex leading into phosphorylation of TBK1 and IRF3, nuclear translocation of p-IRF3 and subsequent IFN-β production during KSHV or HSV-1 de novo infection. Viral genome recognition by IFI16-BRCA1 results in acetylation of IFI16 which interacts with ASC and procaspase-1 in the nucleus. This BRCA1-IFI16-ASC-procaspase-1 complex is transported to the cytoplasm via Ran-GTP resulting in caspase-1 formation, pro-IL-1β cleavage and IL-1β formation [ , ]. These studies demonstrate that the innate nuclear foreign viral genome sensing responses are mediated by two IFI16 complexes, with recognition by the IFI16-H2B-BRCA1 complex resulting in IFN-β responses while recognition by the IFI16-BRCA1 complex results in inflammasome-IL-1β responses.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques: Infection, Western Blot, Control, Translocation Assay, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

doi: 10.1371/journal.ppat.1005967

Figure Lengend Snippet: List of antibodies used in this study.

Article Snippet: BRCA1 (GTX70111) , Mouse monoclonal , GeneTex, Irvine, CA.

Techniques:

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