Journal: EBioMedicine
Article Title: Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy
doi: 10.1016/j.ebiom.2018.11.038
Figure Lengend Snippet: ERα regulation of GJA1 in hTERT-HM cells. RNA and protein extracts were isolated after 96 h of infection with ERα shRNA lentivirus that targets the 3′UTR of ERα mRNA. Representative western blot (A) and densitometric analysis demonstrate a decline in the nuclear ERα66 (B), ERΔ7 isoforms (C) and ERα46 isoforms (D) and the cytoplasmic GJA1 (E) in the hTERT-HM cells upon lentivirus-mediated knockdown of ERα compared to non-silencing pGIPZ shRNA lentiviral control. A significant decline in ERα66/ERα46 (F) and ERΔ7 isoform (G) mRNA levels were also observed in the hTERT-HM cells upon lentivirus-mediated knockdown of ERα compared to non-silencing pGIPZ shRNA lentiviral control. GAPDH and Histone H3 are cytoplasmic and nuclear loading controls. Gene expression was normalized to Rplp0 . *p < 0.05 by two-tailed Student's unpaired t -test, each experiment was performed in triplicate with n = 3 per group. Data shown are mean ± SEM. See also Fig. S3.
Article Snippet: Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20, and then probed with the following primary antibodies: anti- ERα HC-20 (1:200; sc-543; carboxy terminus), anti-ERα G-20 (1:250; sc-544; hinge region), anti-ERα H-184 (1:250; sc-7207; amino terminus) anti-hnRNPG (RBMX) (1:500; sc-48,796) from Santa Cruz Biotechnology, anti-ERα D8H8 (1:1000; #8644; carboxy terminus), anti-ERα D6R2W (1:1000; #13258; amino-terminus) from Cell Signaling Technology, anti-ERα (1:100; ab16660; carboxy terminus) from Abcam and GJA1 (1:1000; C6219) from Sigma-Aldrich.
Techniques: Isolation, Infection, shRNA, Western Blot, Expressing, Two Tailed Test