primary polyclonal anti hif1a antibodies  (Novus Biologicals)

Journal: Oncology Reports

Article Title: M2 macrophages reduce the radiosensitivity of head and neck cancer by releasing HB-EGF

doi: 10.3892/or.2020.7628

Figure Lengend Snippet: In CAL27 cells, inhibition of EGFR by small molecule inhibitor results in reduced NHEJ repair capacity. (A) CAL27 and CAL27 with HB-EGF (50 ng/ml) were assessed by the western blot analysis with anti-EGFR and anti-p-EGFR antibodies. (B) EGFR and EGFR phosphorylation expression in CAL27 cells with or without HB-EGF after 4 Gy radiation. NC, CAL27 cells. (C) Representative images of the number of the p-DNA-PK foci per cell in 5 h after irradiation (4 Gy) in CAL27 cells with or without the EGFR inhibitor; magnification, ×200 (green, p-DNA-PK staining; blue, DAPI). (D) Representative images of the number of the RAD51 foci per cell in 5 h after irradiation (4 Gy) in CAL27 cells with or without the EGFR inhibitor; magnification ×200 (green, p-DNA-PK staining; blue, DAPI). *P<0.05. In CAL27 cells, inhibition of EGFR by small molecule inhibitor results in reduced NHEJ repair capacity. (E-a) Number of p-DNA-PK foci of NHEJ specific markers in CAL27 cells with or without EGFR inhibitors; (E-b) Number of RAD51 foci of HR specific markers in CAL27 cells with or without EGFR inhibitors. (F) IHC analyses of EGFR in cancer and normal tissues; magnification, ×400 (HPV + T, HPV-positive tumor tissues; HPV-T, HPV-negative tumor tissues; HPV + /HPV − N, head and neck normal tissues). (G) EGFR IHC score. HPV-positive cancer tissues (n=19), HPV-negative cases (n=33). (H) Expression of EGFR between HPV-positive (n=98) and HPV-negative (n=420) HNSCC in the TCGA database. Results are presented as the mean ± SD. *P<0.05. HPV, human papilloma virus; HNSCC, head and neck squamous cell carcinoma; EGFR, epidermal growth factor receptor; NHEJ, non-homologous end-joining; HB-EGF, heparin-binding epidermal growth factor; p-, phosphorylated; DNA-PK, DNA-dependent protein kinase; RAD51, RAD51 recombinase; IHC, immunohistochemistry; TCGA, The Cancer Genome Atlas.

Article Snippet: Samples were incubated with the γ H2A histone family member X (H2AX) mouse monoclonal antibody (product code ab26350; Abcam) at 1:500 dilution, primary rabbit polyclonal antibodies against RAD51 recombinase (RAD51; product code ab133534; Abcam) at 1:200, and phosphorylated (p)-DNA-dependent protein kinase (DNA-PK; phosphor S2056; product code ab124918; Abcam) at 1:200 and EGFR inhibitor gefitinib (ZD1839; cat. no. HY-50895; MedChemExpress) overnight at 4°C.

Techniques: Inhibition, Western Blot, Expressing, Irradiation, Staining, Non-Homologous End Joining, Binding Assay, Immunohistochemistry

Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

Article Title: BRCA2 protects mammalian cells from heat shock.

doi: 10.1080/02656736.2017.1370558

Figure Lengend Snippet: Figure 1. RAD51 foci formation after heat shock. (A) Typical photographs of RAD51 in V79 cells at indicated time points after heat shock (44 C for 60 min). a–d, RAD51; e–h, DAPI; i–l, merge. (B) RAD51 foci formation at indicated times after heat shock. Columns show the mean of three independent experiments. Bars indicate the SD. (C) Immunofluorescence staining for cH2AX and RAD51 foci formation in nuclei by DAPI of V79 cells at 4 h after heat shock (44 C for 60 min) and irradiation (X-rays, 10 Gy).

Article Snippet: Then the cells were permeabilised for 5min at 4 C in 0.2% Triton X-100, and blocked in PBS with 1% Bovine Serum Albumin (BSA) for 1 h at 37 C. Cells were then incubated with anti-phospho-H2AX (Ser 139) mouse monoclonal antibodies (Upstate Biotechnology, Lake Placid, NY) at a 1:300 dilution, or rabbit polyclonal anti-RAD51 primary antibodies (H-92, Santa Cruz) for 1 h at room temperature at a 1:300 dilution in PBS containing 1% BSA.

Techniques: Immunofluorescence, Staining, Irradiation

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 2. RAD51 protein levels are elevated in protein extracts from FA fibroblasts of complementation groups A, C, and G. A, RAD51 protein levels were examined by Western blot analysis of equal amounts of nuclear protein extracts prepared from normal diploid fi- broblasts (N) and patient-derived FA fibroblasts of complementation groups A, C, G, and D2. B, RAD51 Western blot analysis was performed on equal amounts of nuclear extracts prepared from normal diploid fibroblasts (PD.792.F (lane 1), PD.751.F (lane 2), PD.715.F (lane 3), PD.793.F (lane 4), CCD-1059Sk (lane 5), CCD-1056Sk (lane 6), and CCD-1108Sk (lane 7)) and HT1080 cells (H). C, the same amounts of the same nuclear protein extracts from A were examined by Western blot analysis for AP endonuclease-1 protein expression. D, RAD51 Western blot analysis was performed on equal amounts of whole cell protein extracts prepared from normal diploid fibroblasts; patient-derived FA fibroblasts of complementation groups A, C, and G and their retrovi- rally corrected counterparts (Acor, Ccor, and Gcor, respectively); and patient-derived FA fibroblasts of complementation group D2. E, the same amounts of the same whole cell protein extracts from D were examined by Western blot analysis for KU86 protein expression.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Western Blot, Derivative Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 3. RAD51 and MRE11 protein levels are elevated in pro- tein extracts from FA fibroblasts of complementation groups A, C, and G. A, Western blot analysis of equal amounts of whole cell protein extracts from normal diploid fibroblasts (N); patient-derived FA fibroblasts of complementation groups A, C, and G and their retrovi- rally corrected counterparts (Acor, Ccor, and Gcor, respectively); and patient-derived FA fibroblasts of complementation group D2 was per- formed by probing the blot with both anti-RAD51 and anti-MRE11 antibodies. B, the same amounts of the same whole cell protein extracts from A were examined by Western blot analysis for AP endonuclease-1 expression.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Western Blot, Derivative Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 4. Normal diploid fibroblasts expressing a dominant-negative FANCC gene have elevated RAD51 protein and high HR activity. A, RAD51 protein (RAD51p) levels were ex- amined by Western blot analysis of equal amounts of whole cell protein extracts prepared from normal diploid fibroblasts that were unmodified (N) or that overex- press the pL554P FANCC gene (NpL554P) and from FA fibroblasts from patients of complementation group C and their retrovirally corrected counter- parts (Ccor). Protein levels were quanti- tated in three independent experiments and are expressed as the -fold increase ver- sus unmodified normal diploid extracts. Er- ror bars represent S.E. *, p 0.05. B, HR frequency was examined in these same cells. Results are an average of at least three independent experiments. Error bars represent S.E. *, p 0.05.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Expressing, Dominant Negative Mutation, Activity Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 5. Cells from a mouse model of FA display elevated RAD51 protein and high levels of HR activity. A, left panel, Western blot analysis of RAD51 protein levels was performed on equal amounts of whole cell protein extracts prepared from embryonic fibroblasts derived from wild-type mice (N) and from mice homozygous for an inactivating deletion of the murine FancC gene (C); right panel, Coomassie Blue- stained gel of the same extracts. B, HR frequency was examined in these same cells. Results are an average of at least four independent experiments in each cell strain. Error bars represent S.E. *, p 0.05 compared with wild-type embryonic fibroblasts in the absence of antibody.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Activity Assay, Western Blot, Derivative Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 6. Protein levels of the RAD51 paralogs are not altered in FA fibroblast extracts. Equal amounts of whole cell protein extracts prepared from FANCC fibroblasts (C) and retrovirally corrected FANCC fibroblasts (Ccor) were examined by Western blot analysis using polyclonal anti-RAD51B, anti-RAD51C, anti-RAD51D, anti-XRCC2, and anti-XRCC3 antibodies.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Western Blot