Ab175245

Immunohistochemistry (IHC) analyses were performed as described previously [21 (link)]. Antibodies against AR (ab9474; dilution, 1:200), PDEF (ab197375; dilution, 1:200), MAD1 (ab175245; dilution, 1:200) and MYC (ab32072; dilution, 1:200) were purchased from Abcam. Anti-Ki67 antibody (sc-23,900; dilution, 1:200) was purchased from Santa Cruz Biotechnology. Normal breast tissue sections were processed simultaneously and were used as positive controls for AR and PDEF, and normal goat serum-substituted primary antibodies were used as negative controls. Two senior pathologists independently quantified IHC slides. IHC scores of PDEF were used to the multiplied result of percentage positivity and staining intensity in the stained tissue area, and total scores ranged from 0 to 6. Percentage positivity was scored as 0 (0–25%), 1 (26–50%) and 2 (> 50%), and staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). A total score of ≥0 and ≤ 3 indicated negative PDEF expression, and a total score of ≥4 indicated positive PDEF expression [21 (link)]. AR expression was considered to be positive if nuclear staining was observed in > 10% tumour cells.

Western blotting was performed as described previously [21 (link)] by using the following primary antibodies: anti-AR antibody (ab9474; dilution, 1:3000), anti-PDEF antibody (ab53881; dilution, 1:1000; Abcam), anti-MAD1 antibody (ab175245; dilution, 1:3000), anti-MYC antibody (ab32072; dilution, 1:3000), anti-β-catenin antibody (ab32572; dilution, 1:3000; Abcam), anti-AKT antibody (sc-135,829; dilution, 1:3000; Santa Cruz Biotechnology), anti-phosphorylated AKT antibody (anti-p-AKT; sc-7985-R; dilution, 1:3000; Santa Cruz Biotechnology), anti-ERK antibody (sc-514,302; dilution, 1:3000; Santa Cruz Biotechnology), anti-phosphorylated ERK antibody (anti-p-ERK; sc-81,492; dilution, 1:3000; Santa Cruz Biotechnology) and anti-EGFR antibody (ab52894; dilution, 1:3000; Abcam).