rabbit anti human 53bp1 polyclonal antibody  (Novus Biologicals)

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: Journal of Biological Chemistry

Article Title: Binding of TPP1 Protein to TIN2 Protein Is Required for POT1a,b Protein-mediated Telomere Protection

doi: 10.1074/jbc.m114.592592

Figure Lengend Snippet: FIGURE 3. DNA damage response in TIN2TPP1-expressing cells is ATR-dependent. A, IF of 53BP1 (red) and telomeric FISH (green) in TIN2F/F MEFs (72 h post-Cre) expressing an EV or the indicated FLAG-HA2 TIN2 alleles. DNA was stained with DAPI (blue). B, quantification of TIFs in TIN2F/F MEFs transduced with an empty vector or TIN2TPP1 (shown together in the top panel) and wild-type TIN2 (bottom panel) as scored for 53BP1 TIFs per nucleus and TIF-positive nuclei (10 53BP1 foci at telomeres) (n 200) after Cre (72 h). Data are mean S.D. from at least three independent experiments. **, p 0.05 (paired Student’s t test); ns, not significant. C, immunoblots for Chk1 and Chk2 phosphorylation in TIN2F/F MEFs expressing the indicated FLAG-HA2 TIN2 alleles and treated with Cre (72 h) as indicated. -tub, -tubulin. D, IF of 53BP1 (red) and telomeric FISH (green) in TIN2F/F MEFs (72 h post-Cre) expressing EV or TIN2TPP1 with or without ATM inhibitor (ATMi) and ATRi. DNA was stained with DAPI (blue). E, quantification of TIF-positive nuclei (10 53BP1 foci at telomeres; n 200) in TIN2F/F MEFs transduced with an EV or TIN2TPP1 and treated with ATM inhibitor and ATRi after Cre (72 h) as in D. Data are mean S.D. from three independent experiments. **, p 0.05 (paired Student’s t test).

Article Snippet: Indirect Immunofluorescence—Cells were grown on coverslips and fixed with 2% paraformaldehyde in PBS for 10 min. Telomeric DNA FISH was combined with IF as described previously (8) using the following antibodies: polyclonal rabbit anti-TRF1 (catalog no. 644), polyclonal rabbit anti-mTRF2 (catalog no. 1254), polyclonal rabbit anti-mRap1 (catalog no. 1252), and polyclonal rabbit anti-human 53BP1 antibody (catalog no. NB 100-304, Novus); mouse monoclonal anti-MYC antibody (catalog no. 9E10, Sigma); and anti-HA antibody (catalog no. 12CA5, Roche).

Techniques: Expressing, Staining, Transduction, Plasmid Preparation, Western Blot, Phospho-proteomics