Ab14686

Tissue microarray of TNBC patients with information of clinical–pathological parameters was purchased from Outdo Biotech (HBreD090Bc01; Shanghai, China). Paraffin‐embedded sections of xenograft tissues were subjected to deparaffinization and rehydration. H/E staining of sections was carried out using H/E staining kit (Beyotime, Shanghai, China) according to manufacturer’s instructions. For immunohistochemical staining of tissue microarray and sections of xenograft and antigen retrieval, blocking of non‐specific binding and incubation of primary antibodies at 4 °C overnight was sequentially conducted. The primary antibodies used were list as follows: anti‐phospho‐EGFR (ab40815; Abcam, 1 : 200) and anti‐SGLT1 (ab14686; Abcam, 1 : 100). After incubation with secondary goat anti‐rabbit immunoglobulin conjugated to peroxidase‐labelled dextran polymer (SV0002; Boster) at 37 °C for 1 h, visualization, counterstaining with haematoxylin and mounting were performed. Semiquantitative evaluations of protein expression were scored on the basis of the intensity and the percentage of phospho‐EGFR‐ or SGLT1‐positive tumour cells as previously described (Wang et al., 2014).

Tissue lysates were prepared using Cell Lysis Buffer for Western and IP (Biotime Biotechnology, Xiamen, Fujian, PRC). Protein concentrations were detected using a Enhanced BCA Protein Assay Kit (Biotime Biotechnology). Equal amounts of proteins from each sample were subjected to SDS-PAGE followed by a transfer of proteins to nitrocellulose membranes. Membranes were blocked with a non-protein blocking solution (Sangon Biotech) and incubated with a primary antibody overnight at 4°C. After washing with TBST, membranes were incubated with a secondary antibody linked to Horseradish peroxidase (HRP). The blots were then developed with an ECL detection system as per the manufacturer's instructions. Rabbit anti-SGLT1 (ab14686), anti-PEPT1 (ab203043), anti-P47^phox (ab74095), anti-Nrf2 (ab62352), anti-Keap1 (ab139729), anti-iNOS (ab178945), anti-MR (ab229987), anti-p-IRF3 (Ser396)(ab138449), and anti-IRF3 (ab25950) monoclonal antibodies were obtained from Abcam. Rabbit anti-Arg (93668S), anti-pSTAT1 (Tyr701) (9167S), anti-STAT1 (9172T), anti-STAT6 (5397S), anti-STAT3 (30835S) monoclonal antibodies were obtained from CST (Shanghai, PRC). Mouse anti-β-actin monoclonal antibody was obtained from Biotime Biotechnology. The relative band density was determined using ImageJ software.

Protein was extracted from adrenal tissue for Western blot. The protein lysates were separated in 10% SDS-PAGE and transferred to PVDF membrane. PVDF membrane was closed at room temperature with 5% skimmed milk dissolved in Tris-buffered saline with Tween-20 (TBST) for 1 h, followed by incubation of the primary antibody in a 4 °C overnight. The next day, after washing the membrane with TBST for 5 min ×3, the membrane was incubated with secondary antibody for 1 h at room temperature. After the incubation was completed, the membranes were washed again 3 times and developed by ECL method. The primary antibodies and dilution ratios used in this study were as follows: anti-SGLT2 (1:1000, ab14686, abcam), anti-SGLT2 (1:1000, ab37296, abcam), GAPDH (1:5000, 60004-1-Ig, proteintech).