Epr17573

The IHC staining was performed on 4 μm tissue microarrays sections using a Novolink polymer detection system (RE7150-K, Leica Biosystems, Newcastle, UK), as previously described [12 (link)]. Evaluation of membranous staining for SLC7A5 1:200 (EPR17573, Abcam, Cambridge, UK) and SLC3A2 1:2000 (HPA017980, Sigma-Aldrich, Dorset, UK) were based on a semi-quantitative assessment of invasive tumour cells using a modified H-score as previously described [11 (link),12 (link)]. TMA cores were only assessed if the tumour burden was >15%.

The antibody specificity of anti-SLC7A5 (EPR17573, Abcam, UK) was validated using western blotting in human embryonic kidney (HEK) 293 T over expression lysate (Origene Technologies, Rockville, MD, USA) and MDA-MB-175 (luminal B-like), T47D and MCF7 (luminal A) [19 (link)] breast cancer lysate (American Type Culture Collection; Rockville, MD, USA). A dilution of 1:200 of the primary antibody and 1:1000 HRP-conjugated (Dako) secondary antibodies were applied: 5% milk /PBS-Tween (0.1%) (Marvel Original Dried Skimmed Milk, Premier Food Groups Ltd., UK) was used for blocking. Mouse monoclonal anti-β-actin primary antibody was used as a marker of endogenously expressed control. SLC7A5 bands were visualised using enhanced chemiluminescence (ECL) showing a single specific band at the correct predicted size (40 kDa) for the SLC7A5 protein.