Antibody-based Protein Quantification in Cellular Studies

Antibodies for β-actin (sc-47778), COX2 (sc-19999), MMP1 (sc-21731), NRF1 (sc-101102), and SOD2 (sc-30080) were obtained from Santa Cruz Biotechnology. Antibodies for acetyl-histone H4 K5, K8, K12, and K16 (H4K5,8,12,16ac, #PA5-40084) were obtained from Invitrogen. Antibodies for anti-histone H3 acetyl K9, K14, K18, K23, K27 (H3K9, 14, 18, 23, 27ac, ab47915), ERα (ab3575), ERβ (ab3576), H4K20me1 (ab9051), H4K20me3 (ab9053), H4R3me1 (ab17339), H3K9me2 (ab1220), H3K9me3 (ab8898), H3K27me2 (ab24684) and H3K27me3 (ab6002), H2AX (ab20669), and γH2AX (ab2893) were obtained from Abcam. The antibody for 8-oxo-dG (4354-MC-050) was obtained from Novus Biologicals. The 3-nitrotyrosine (3-NT) was measured using 3-Nitrotyrosine ELISA Kit (ab116691 from Abcam) per manufacturers’ instructions. The mitochondrial fraction was isolated using a Pierce Mitochondria Isolation Kit (Pierce Biotechnology) according to manufacturers’ instructions. The protein concentration was measured using the Coomassie Protein Assay Kit (Pierce Biotechnology) per manufacturers’ instructions. Luciferase activity assay was carried out using the Dual-Luciferase™ Assay System (Promega) and the transfection efficiency was normalized using a cotransfected renilla plasmid (23 (link)). 17β-estradiol (E2, #E2758) and TNFα (#T0157) were obtained from Sigma.

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Xiang D., Zhao M., Cai X., Wang Y., Zhang L., Yao H., Liu M., Yang H., Xu M., Li H., Peng H., Wang M., Liang X., Li L, & Yao P. (2020). Betulinic Acid Inhibits Endometriosis Through Suppression of Estrogen Receptor β Signaling Pathway. Frontiers in Endocrinology, 11, 604648.

Publication 2020

(sc-30080) (ab3576 ab9051), ab9053), ab17339 ab24684 ab6002 3-Nitrotyrosine ELISA Kit Pierce Mitochondria Isolation Kit Coomassie Protein Assay Kit Dual-Luciferase™ Assay System 17β-estradiol (E2, #E2758)

17β estradiol 3 nitrotyrosine 8 oxo dg Actin Anti antibodies Antibodies Antibody Assay Biologicals Cox2 Elisa Histone h3 Histone h4 Isolation Luciferase Mitochondria Mmp1 Novus Plasmid Promega Protein Renilla Sod2 Tnfα Transfection

Corresponding Organization : Hainan Maternal and Child Health Hospital

Other organizations : Wuhan City Chinese Medicine Hospital

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Variable analysis

independent variables

17β-estradiol (E2)
TNFα

dependent variables

Antibody expression for β-actin, COX2, MMP1, NRF1, SOD2
Acetylation of histone H4 at K5, K8, K12, K16
Acetylation of histone H3 at K9, K14, K18, K23, K27
Expression of ERα and ERβ
Methylation of histone H4 at R3, K20
Methylation of histone H3 at K9, K27
Expression of H2AX and γH2AX
Levels of 8-oxo-dG
Levels of 3-nitrotyrosine

control variables

Protein concentration measured using Coomassie Protein Assay Kit
Luciferase activity assay normalized using cotransfected renilla plasmid

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