Sc 815

Fixed tissues were dehydrated using a full-automatic dehydrator, paraffin embedded and then sectioned into 5-mm thick samples. First, the dewaxed sections were placed into the dyeing tank with 3% methanol hydrogen peroxide at room temperature for 10 min. The samples were rinsed with PBS three times for 5 min each. The slices were dipped into 0.01 M citrate buffer (pH 6.0) and then heated in the microwave until boiling for 5 min interval; the heating was repeated once more. After cooling, the slice was washed with PBS two times for 5 min each. The sections were then blocked with a blocking serum (ZLI-9021, ZSGB-BIO) at room temperature for 20 min. The sections were incubated with the AR (C-19) antibody (1:100; sc-815, Santa Cruz) at 4°C overnight and then with a secondary antibody (1:250; sp-9001, ZSGB-BIO) for 30 min at 37°C. Samples were rinsed with PBS three times for 5 min each and then processed with the Concentrated DAB kit (K135925C, ZSGB-BIO). Sections were then dehydrated in alcohol, cleared in xylene and mounted in synthetic resin. Mitochondria staining of paraffin tissue sections was carried out using the anti-prohibitin antibody mitochondrial marker (1:100; ab28172, Abcam). The mean density of immunohistochemical staining was measured using Image-Pro Plus (IPP). n = 4 per group; five technical replicates.

Total protein was extracted from flow-sorted cells using T-PER Tissue Protein Extraction Reagent (Fisher), separated by SDS-PAGE and transferred onto PVDF membrane according to standard protocols. Membranes were probed with antibodies directed against AR (sc-815, Santa Cruz Biotechnology, 1:500) and β-actin (sc-47778, Santa Cruz Biotechnology, 1:500). Signal was visualized with secondary HRP conjugated antibodies and Clarity Western ECL Substrate (Biorad). Full size images are presented in Supplementary Fig. 12.