Sc 10790

Luminal A type breast cancer tissue array slides (BR1507) were purchased from US Biomax. For PLA of breast cancer tissues, slides were deparaffinized, processed for antigen retrieval, and then subjected to PLA using antibodies against IFI16 (sc-8023, Santa Cruz Biotechnology), HIF1α (sc-10790), and PRMT2 (ab154154, Abcam).

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.

After whole tubule pellet lysis with 450 mmol/L NaCl, 50 mmol/L Tris‐EDTA pH 8, 10% glycerol, 0.5% NP40 and inhibitors of proteases and phosphatases, VEGF was analyzed by western blotting using the pan‐VEGF antibody from Santa Cruz Biotechnology (Heidelberg, Germany) (sc‐152) and the anti‐VEGFxxxb antibody from ABCAM (Cambridge, UK) (ab14994). FLK1, TIE‐2, GLUT1, Aldolase A and Tubulin were analyzed by western blotting with antibodies from, respectively, Cell Signaling (2479), Santa Cruz (sc‐9026), ABCAM (ab32551), Cell Signaling (Danvers, MA) (3188), and Calbiochem (Millipore SAS, Molsheim, France) (CP06). HIF1A, HIF2A, and actin (epitope present in the six isoforms of vertebrate actin) were determined in nuclear fraction after tubule lysis (Zou et al. 2001 (link)) by western blotting with antibodies from, respectively, Santa Cruz Biotechnology Inc (sc‐10790), ABCAM (ab8365) and Chemicon (Millipore SAS) (MAB1501) as previously described (Hervouet et al. 2005 (link)). Protein levels from the blots were evaluated using the gel analysis ImageLab software (BioRad, Hercules, CA) and the ratio of protein levels in the normal glucose supply versus the low and high glucose supplies was represented as fold change.