rabbit anti human 53bp1  (Bethyl)

Journal: Frontiers in Microbiology

Article Title: Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production

doi: 10.3389/fmicb.2021.659028

Figure Lengend Snippet: Influence of HO-1 induction on p38 MAPK activation in BeWo cells under T. gondii infection. (A,B) BeWo (1 × 10 6 cells/2,000 μl/well) cells were cultured in six-well plate during 24 h, infected with T. gondii (1:1) or not (control) for 3 h, treated with hemin (80 μM) or not (NaOH at 0.08% as vehicle) during 24 h, and submitted to western blotting assays for detection of phosphorylated p38 MAPK and β-actin. Protein bands were photodocumented, and densitometric analysis was performed by the ratio among phosphorylated p38 MAPK ( p -p38 MAPK) and its corresponding β-actin (endogenous control). (A) Representative blots for p -p38 MAPK and β-actin in the non-infected (NI) or infected ( T. gondii ) and hemin-treated or not (vehicle) BeWo cells. (B) Densitometric analysis for p -p38 MAPK. (C) BeWo (3 × 10 4 cells/200 μl/well) cells were cultured in 96-well plate during 24 h, pretreated with 10 μM of SB203580 (p38 MAPK inhibitor) or not (control) for 3 h, infected with T. gondii (1:1) for 3 h, and treated with hemin (80 μM) or not (control) during 24 h, and the T. gondii intracellular proliferation was analyzed by the β-galactosidase assay. The data were shown as number of tachyzoites (× 10 3 ). For both assays, the results were expressed as mean ± SEM from two or three independent experiments performed in six or eight replicates. Significant differences were statistically assessed by the Mann-Whitney test (B) or by one-way ANOVA with Bonferroni’s posttest (C) ; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Then, the membranes were blocked for 1 h with blotting buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% ( v / v ) Tween 20, and pH 7.4) added with 4% non-fat dry milk (Molico, Nestlé ® , São Paulo, SP, Brazil) and incubated, overnight, with the following primary antibodies diluted in a blotting buffer with 2% non-fat dry milk: goat polyclonal anti-human-HO-1 (1:1,000; Santa Cruz Biotechnology Cat# sc-1797, Santa Cruz, CA, United States), rabbit polyclonal anti-human-phospho-p38 MAPK (T 180 /Y 182 ) (1:400; R&D Systems Cat# AF869, Minneapolis, MN, United States), or mouse monoclonal anti-human-β-actin (1:1,000; Santa Cruz Biotechnology Cat# sc-81178).

Techniques: Activation Assay, Infection, Cell Culture, Control, Western Blot, MANN-WHITNEY

Journal: Frontiers in Microbiology

Article Title: Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production

doi: 10.3389/fmicb.2021.659028

Figure Lengend Snippet: Proposed mechanisms triggered by the HO-1 to control T. gondii infection by BeWo trophoblast cells. (A) Upper panel, a non-infected BeWo cell expresses normal levels of HO-1 and phosphorylated p38 MAPK, in addition to secreting baseline levels of IL-6. (B) Left panel, when BeWo cells were infected with T. gondii , the amounts of HO-1 and phosphorylated p38 MAPK were significantly decreased and the IL-6 levels were upregulated. (C) Right panel, when finally T. gondii -infected BeWo cells were treated with hemin, a specific HO-1 inducer, the parasite proliferation was significantly reduced, sequentially associated with an overexpression of HO-1, an increasing of the p38 MAPK phosphorylation and a releasing of high IL-6 levels, which was higher than those naturally secreted by the T. gondii -infected cells non-induced for HO-1 (left panel).

Article Snippet: Then, the membranes were blocked for 1 h with blotting buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% ( v / v ) Tween 20, and pH 7.4) added with 4% non-fat dry milk (Molico, Nestlé ® , São Paulo, SP, Brazil) and incubated, overnight, with the following primary antibodies diluted in a blotting buffer with 2% non-fat dry milk: goat polyclonal anti-human-HO-1 (1:1,000; Santa Cruz Biotechnology Cat# sc-1797, Santa Cruz, CA, United States), rabbit polyclonal anti-human-phospho-p38 MAPK (T 180 /Y 182 ) (1:400; R&D Systems Cat# AF869, Minneapolis, MN, United States), or mouse monoclonal anti-human-β-actin (1:1,000; Santa Cruz Biotechnology Cat# sc-81178).

Techniques: Control, Infection, Over Expression, Phospho-proteomics

Journal: Frontiers in Public Health

Article Title: In vitro Assessment of the DNA Damage Response in Dental Mesenchymal Stromal Cells Following Low Dose X-ray Exposure

doi: 10.3389/fpubh.2021.584484

Figure Lengend Snippet: DNA double strand break formation and repair kinetics. (A) Dental pulp stromal cells from deciduous teeth show a significantly increased number of DNA double strand breaks following irradiation with 50 and 100 mGy 30 min and 1 h after radiation exposure. (B) The number of co-localized foci, observed in stromal cells from the apical papilla after exposure to 100 mGy, was significantly increased compared to 0 mGy 30 min, 1 and 4 h after irradiation ( P < 0.0001, P < 0.0001, P = 0.0267, respectively). 50 mGy irradiated samples showed more foci 30 min and 1 h p.i ( P = 0.0018, P = 0.0004, respectively). (C) In dental follicle stromal cells, more foci were observed 30 min, 1 and 4 h after exposure to 100 mGy ( P < 0.0001, P < 0.0001, P = 0.0374, respectively). Thirty minutes and one hour after exposure to 50 mGy and 30 min after exposure to 20 mGy the amount of co-localized foci was increased as well in DFSC ( P < 0.0001, P = 0.0015, P = 0.0030, respectively). The number of foci returns to control levels 24 h after irradiation. (D–G) Representative image from stromal cells from the apical papilla taken 60 min after irradiation with 100 mGy. The nucleus (D) shows five clear γH2AX (E) and 53BP1 (F) foci, which co-localize (G) . * P ≤ 0.05; ** P ≤ 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies were diluted in TNB, the mouse anti-human γH2AX monoclonal antibody (05-636, Millipore, Massachusetts, USA) was diluted 1:300 and the rabbit anti-human 53BP1 polyclonal antibody (NB100-304, Novus Biological, Abingdon, UK) was diluted 1:1,000.

Techniques: Irradiation, Control

Journal: Frontiers in Public Health

Article Title: In vitro Assessment of the DNA Damage Response in Dental Mesenchymal Stromal Cells Following Low Dose X-ray Exposure

doi: 10.3389/fpubh.2021.584484

Figure Lengend Snippet: Linear dose response relationship of co-localized γH2AX and 53BP1 foci in dental stromal cells.

Article Snippet: Primary antibodies were diluted in TNB, the mouse anti-human γH2AX monoclonal antibody (05-636, Millipore, Massachusetts, USA) was diluted 1:300 and the rabbit anti-human 53BP1 polyclonal antibody (NB100-304, Novus Biological, Abingdon, UK) was diluted 1:1,000.

Techniques: Irradiation, Significance Assay

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: (A) Spontaneous frequency of phospho-53BP1 foci in non-stimulated and unirradiated PBLs of five volunteers (V1-V5). Error bars signify SD (5 × 3 i.e., five individuals with triplicate blood sampling). More than 300 PBLs were analyzed for estimating the spontaneous frequency of phospho-53BP1 in each volunteer. (B) Representative PBLs with (i) no foci (ii) one focus (iii) two foci (iv) numerous foci (could be apoptotic cell).

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Sampling

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: (A) Phospho-53BP1 foci, decay kinetics, in the blood sample of five volunteers [two females (with age 22 and 24 y) and three males (with age 25 y, 31 y and 35 y)] after irradiation with 1, 2, 3, and 4 Gy. Error bars represents SEM ( n = 5 × 3, i.e., five volunteers with triplicate sampling). Number of cells analyzed/scored > 300/dose/time point/volunteer. (B) Illustration of the disappearance of phospho-53BP1 foci at 1, 2, 4, 8, 16, and 24 h of incubation after irradiation with 1, 2, 3, and 4 Gy. Dispersion of the number of phospho-53BP1 foci in PBLs irradiated with 1 Gy (cumulative data of all five volunteers) after incubation of (C) 1 h (D) 2 h (E) 4 h (F) 8 h (G) 16 h, and (H) 24 h.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Irradiation, Sampling, Incubation, Dispersion

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Fitting parameters for phospho-53BP1 foci decay for doses 1, 2, 3, and 4 Gy up to 24 h of incubation after irradiation.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Incubation, Irradiation

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Half-lives of phospho-53BP1 foci for doses of 1, 2, 3, and 4 Gy estimated using the formula T1/2 = 0.693*t1.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques:

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Dose-response curves for 60 Co-γ-rays-induced-phospho-53BP1 foci formation post-irradiation incubation of (A) 1 and 2 h (B) 4 and 8 h (C) 16 and 24 h. (D) Estimated minimum detection limits of the dose-response curves established at 1, 2, 4, 8, 16, and 24 h of incubation, following ISO recommendations (3 sigma of the background frequency) (E) Variation of the slope (53BP1 foci/cell/Gy) of the established dose-response curves, generated at 1, 2, 4, 8, 16, and 24 h of incubation, post-irradiation. (F) Microscopic images showing an increasing number of phospho-53BP1 foci in the nucleus of PBLs with increasing doses (0 to 2.0 Gy after 1 h and 3 and 4 Gy after 4 h of incubation, post irradiation). (G) Representative images of monocytes (kidney-shaped nucleus) with and without 53BP1 foci (these cells were excluded from the scoring of 53BP1 foci).

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Irradiation, Incubation, Generated

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: (A) Colocalization of phospho-53BP1 and γH2AX foci in the nucleus of human lymphocyte exposed with various doses (at 1 h incubation post irradiation). Correlations established between dose-response curves generated for phospho-53BP1 foci and γH2AX foci (data not shown) at dose points (B) 0.05 Gy (C) 0.1 Gy (D) 0.25 Gy (E) 0.5 Gy (F) 1 Gy (G) 2 Gy (H) 4 Gy with varying incubation time points (1–24 h) after irradiation. Correlations were established between dose-response curves generated for phospho-53BP1 foci and γH2AX foci, at (I) 1 h (J) 2 h (K) 4 h (L) 8 h (M) 16 h and (N) 24 h of incubation after irradiation with various doses (0–4 Gy).

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Incubation, Irradiation, Generated

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Graphical illustration of the estimated doses of blinded samples (Z1–Z6) by immunofluorescence (phospho-53BP1 and γH2AX) and cytogenetic (dicentric and reciprocal translocation) assays.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Immunofluorescence, Translocation Assay

Journal: Frontiers in Public Health

Article Title: Establishment of in vitro Calibration Curve for 60 Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays

doi: 10.3389/fpubh.2022.845200

Figure Lengend Snippet: Comparative evaluation of established response curve (phospho-53BP1) by estimating biological doses of 6 dose blinded samples by phospho-53BP1, γH2AX, dicentric, and translocation assays.

Article Snippet: A total of 50 μl of rabbit anti-phospho 53BP1 human monoclonal IgG (primary antibody; Cell Signaling Technology, Massachusetts, USA) with a dilution of 1:200 (in PBS with 2% FCS) was applied on each cell bearing cover glass and incubated for 1 h in a humidified chamber.

Techniques: Translocation Assay, Immunofluorescence