Western blotting was carried out in compliance with the standard protocol. Antibodies used were: p-P53(ser15) (#9286), p-Rb(ser795) (#9301), p-p38MAPK(Thr180/tyr182) (#9212), extracellular signal-regulated kinases 1/2 (#4695), E-cadherin (#3195), β-catenin (#8481), tubulin (#2128) (Cell Signaling Technology, Danvers, MA, USA), GAPDH (ap7873a) and MMP2 (am1844a) (ABGENT, San Diego, CA, USA), DHRS2 (PA5-25258) (Thermo Fisher Scientific), MDM2 (M4308) (Sigma-Aldrich) and 8-oxoguanine (AB20646) (Abcam, Cambridge, UK). pCDH was bought from System Biosciences (Palo Alto, CA, USA). pCDH-DHRS2-V1 and DHRS2-V2 were constructed and sequenced. pLKO.1-DHRS2-shRNA targeting both variants was bought from Sigma-Aldrich.
8 oxoguanine
Manufactured by Abcam
Sourced in United Kingdom
8-oxoguanine is a compound that is used in research to study oxidative DNA damage. It is a modified guanine base that is formed when guanine is oxidized by reactive oxygen species. 8-oxoguanine can be used as a biomarker to measure oxidative stress in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
P rb ser795, by Cell Signaling Technology (1 mentions)
P p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Mdm2 m4308, by Merck Group (1 mentions)
P p53 ser15, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Gamma h2ax, by Novus Biologicals (1 mentions)
3 3 diaminobenzidine substrate, by Thermo Fisher Scientific (1 mentions)
Polyclonal rabbit anti caspase3, by Abcam (1 mentions)
Entellan, by Merck Group (1 mentions)
Iba 1, by Abcam (1 mentions)
Nfκb p105 50, by Cell Signaling Technology (1 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions)
Hdac4, by Abcam (1 mentions)
γ h2ax, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Phospho jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
4 protocols using 8 oxoguanine
1
Western Blot Protocol for Protein Analysis
2
Immunohistochemical Analysis of Placental Oxidative Damage
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The immunohistochemical detection of 8-Oxoguanine (oxidative DNA damage marker), gamma H2AX (DNA-double strand breaks), APE-1 and OGG1 (DNA repair enzymes), and cleaved caspase-3 (apoptosis) in placental sections from GDM and AnxA1−/− mice was performed. Endogenous peroxide activity was blocked using 3% hydrogen peroxide for 30 min. The tissue sections were then incubated with the following primary antibodies overnight at 4 °C: 8-Oxoguanine (Abcam, Cambridge, UK), gamma H2AX (Novus Biologicals, Littleton, CO, USA), OGG1 (Novus Biologicals), APE-1 (Novus Biologicals), polyclonal rabbit anti-caspase-3 (Abcam). For negative control, sections were incubated with 10% TBS-BSA (Sigma–Aldrich, St. Louis, MO, USA) instead of the primary antibody. After washing with TBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam). Staining was visualized using a 3,3′-diaminobenzidine substrate (Invitrogen, Waltham, MA, USA). Finally, the sections were counterstained with hematoxylin (Inlab Confiança, São Paulo, Brazil) and mounted using Entellan (Merck, Germany).
3
Comprehensive Antibody Panel for Cellular Signaling
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PCNA, p38, phospho-p38 (Thr180/Tyr182), ERK, phospho-ERK (Thr202/Tyr204), JNK, phospho-JNK (Thr183/Tyr185), NFκB p105/50, NFκB p65, phospho-NFκB p65 (Ser536), RelB, Akt, phospho-Akt (Ser473), and phospho-Akt (Thr308) antisera were purchased from Cell Signaling Technology (Danvers, MA, USA); CD45, Iba-1, GFAP, γ-H2AX, HDAC4, MAP2, GAPDH, 8-oxoguanine, and Ki67 antisera were purchased from Abcam (Cambridge, MA, USA); and cyclin A1 and c-Rel antisera were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Secondary antisera conjugated with fluorescent Alexa dye 488 and 647 and Cy3 were purchased from Life Technologies and Jackson ImmunoResearch (West Grove, PA, USA). HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology and Life Technologies.
4
Immunostaining of Myogenic Cells
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For immunostaining, cells grown on plates were fixed with 4% paraformaldehyde at room temperature for 15 min and subsequently permeabilized with 0.3% Triton X-100/ PBS. Cells were blocked in 10% goat serum diluted in 0.1% Triton X-100/ PBS at room temperature for 1 h. Primary antibodies were diluted in 1% goat serum/ 0.1% Triton X-100 and incubated at room temperature for 2 h. Cells were washed and secondary antibodies were diluted in 1% goat serum/0.1% Triton X-100 and incubated at room temperature for 1 h. Cells were stained with Hoechst dye (1:2,000 in PBS) at room temperature for 10 min. Primary antibodies include: Pax3 (DSHB, 1:20), Pax7, (DSHB, 1:20), MyoD (Santa Cruz Biotechnology, sc-377460, 1:100), MyoG (Santa Cruz Biotechnology, sc-12732, 1:100), MYH1 (clone MF20, DSHB, 5μg/ml). MYHC-IIb eFluor 660 (50-6503-32; Thermo Fisher; 1:100), α-actinin (sc-7453; Santa Cruz; 1:500), 8-oxo-guanine (ab206461; Abcam; 1:400). Fusion index was calculated as a ratio of the number of tdTO+ or MuSC nuclei within a multinucleated myotube to the total number of tdTO+ or MuSC nuclei. A minimum of 4 independent microscopic fields was used for each group over three independent differentiation experiments at 24 and 36 hours in DM.
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