Anti erα antibody d8h8

For ERα-immunoprecipitation, HeLa cells (ATTC) were transfected with 3 μg hERα-pSG5 or pSG5 using Lipofectamine^® 2000 (Thermo Scientific) and OptiMEM (Invitrogen). 24 h after transfection, cells were stimulated for 24 h with E2 and/or C18:0, in concentrations as described for THP-1 cells. Cells were lysed with RIPA (with proteinase-inhibitors, Complete Mini, Roche), sonicated (Sonopuls HD 2070, 30 s, 40–50%), and centrifuged. Supernatant was used to perform immunoprecipitation (IP) with anti-ERα-antibody (D8H8, Cell Signaling) bound to Protein A Sepharose-beads (Amersham). For western immunoblotting a distinct anti-ERα-antibody (sc-8002, Santa Cruz) was used. Washed beads-antibody-ERα complexes were analysed for fatty acids- binding as described above.

Cells were homogenized in RIPA lysis buffer for protein extraction, supplemented with protease inhibitors (Thermo Scientific, USA). Denatured proteins were separated in 12% SDS–PAGE and then transferred onto nitrocellulose papers (Pall, USA). After blotting, nitrocellulose papers were incubated with specific antibodies. Primary antibodies: anti-ERα antibody D8H8, 1:2000 (Cell Signaling Technology, USA, #8644); anti-ERα F-10, 1:1000 (Santa Cruz Biotechnology, USA, #sc-8002), anti-ERα 1D5, 1:1000 (Invitrogen, USA, #MA5-13191), anti-ERα-36, 1:200 (Alpha Diagnostic International, USA, #ERA361-A); anti-β-actin 13E5, 1:2000 (Cell Signaling Technology, USA, #8457); anti-HDAC2, 1:1000 (Cell Signaling Technology, USA, #2540); anti-VDAC1 N-18 (Santa Cruz Biotechnology, USA, #sc-8828). Secondary antibodies (HRP conjugated): anti-rabbit, 1:5000 (Cell Signaling Technology, USA, #7074); anti-mouse, 1:2000 (Cytiva, USA, #RPN4201). Immunolabelling was visualized using ECL procedure (PerkinElmer, USA). Uncropped and unprocessed scans of the most important blots are supplied in the Source Data file. All blots derive from the same experiment and they were processed in parallel.

ERα immunostaining (IHC) was performed on Benchmark Ultra using the ultraView DAB Detection kit (Ventana, USA). Antigen retrieval was performed onboard with UltraCC1 buffer (pH 8.2–8.5) at 95 °C for 52 min. Primary antibody (anti-ERα antibody D8H8, Cell Signaling Technology, USA, #8644) 1:100 for 28 min at 37 °C. Secondary antibody 1:100 for 1 h. Images were obtained using a Zeiss Axiovert Widefield Microscope and Zeiss ZEN software (Carl Zeiss, Germany). Immunofluorescence was performed using Leica Bond RX stainer and the Bond Polymer Refine Detection kit (Leica, Germany). Antibodies and detection: OXPHOS (2 µg/ml, Invitrogen, USA, #A-21347); ERα D8H8 (1 µg/ml, Cell Signaling Technology, USA, #8644); Alexa Fluor Tyramide signal amplification reagents (Life Technologies, USA); 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA). Slides were mounted in Mowiol 4–88 (Calbiochem, USA). Confocal imaging was performed on a Leica SP8 inverted microscope (Leica, Germany). Image processing and analysis (2-D and 3-D) was performed using Imaris software (Bitplane, CH).