Western blotting and coimmunoprecipitation were performed as described previously using specific antibodies against aromatase (#677; kindly provided by Dr. Dean P. Edwards (Baylor College of Medicine), and sc-14245, Santa Cruz Biotechnology), IFI16 (sc-8023, Santa Cruz Biotechnology), Actin (sc-1616), HIF1α (sc-10790), PRMT2 (ab154154, Abcam), and Ifi204 (NBP2-27153, Novus Biologicals) [49 (link)]. To detect protein–protein interactions with high selectivity and sensitivity, PLAs were performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma Aldrich) according to manufacturer’s protocol. Briefly, cells were fixed and incubated with primary antibodies against IFI16 (sc-8023, Santa Cruz Biotechnology), HIF1α (sc-10790), and PRMT2 (ab154154, Abcam). The cells were incubated with PLA probes and the subsequent ligation and rolling circle amplification were performed. The PLA signals were visualized using a Zeiss LSM 710 confocal microscope (Carl Zeiss).
The amounts of IFNα, IFNβ, and IFNγ protein were measured using commercial ELISA kits (MBS2506739, MBS2513798, and MBS2512904, respectively, MyBioSource) according to the manufacturer’s protocol. The amount of estradiol was measured using Estradiol ELISA kit (#582251 and #501890, Cayman) according to the manufacturer’s protocol.
The amounts of IFNα, IFNβ, and IFNγ protein were measured using commercial ELISA kits (MBS2506739, MBS2513798, and MBS2512904, respectively, MyBioSource) according to the manufacturer’s protocol. The amount of estradiol was measured using Estradiol ELISA kit (#582251 and #501890, Cayman) according to the manufacturer’s protocol.