Ab106527

Total protein was extracted using RIPA lysis buffer supplemented with EDTA-free Protease Inhibitor Cocktail (#4693132001; Roche, Germany), and were boiled in SDS loading buffer. 10 μg protein was separated by 10% SDS-PAGE, followed by transferring onto 0.45 μm nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in PBST and incubated with primary antibodies against ST3GAL6 (1/500, ab106527; Abcam), GATA3 (1/500, #558686; BD, USA), EGFR (1/1000, #4267; Cell Signaling Technology (CST), USA), phospho-EGFR (Tyr1068) (1/1000, #3777; CST), AKT (pan) (1/1000, #4691; CST), phospho-AKT (Ser473) (1/1000, #4060; CST), p44/42 MAPK (ERK1/2) (1/1000, #4695; CST), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1/1000, #4370; CST), STAT3 (1/1000, #9139; CST), phospho-STAT3 (Tyr705) (1/1000, #9145; CST), or GAPDH (1/1,000, sc-47724; Santa Cruz Biotechnology, USA) at 4℃ overnight. After rinse with PBST three times, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies (1/5,000; Jackson ImmunoResearch, USA; Table S1), followed by the detection with the Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, USA).

Fifty-two patients who initially diagnosed as primary UBC were enrolled and samples were collected after surgery in Shanghai General Hospital (Shanghai, China). This study was approved by the Institutional Review Board of Shanghai General Hospital, with the written informed consent from the corresponding patients. The formalin-fixed paraffin embedded tissues were cut and 5 μm-thick sections were collected on super-frost, positively charged glass slides. IHC was carried out as described previously 32 (link). Briefly, after antigen retrieval by autoclave in citrate buffer at pH 6, sections were incubated with primary anti-ST3GAL6 antibody (1/100, ab106527; Abcam, USA) overnight at 4 °C, followed by the incubation with the horseradish peroxidase (HRP)-conjugated antibody. The slides were visualized by DAB visualization kit (DAB-0031; Maixin_Bio, China), counterstained with hematoxylin. Images were acquired by a slide scanner (NanoZoomer 2.0-HT; HAMMATSU, Japan) and analyzed by NDP Serve slide distribution and management software (HAMMATSU). IHC slides were evaluated by two pathologists independently. The highest intensity was initially scored as 5 and the lowest was scored as 1; and the final score of the ST3GAL6 staining was the multiplication of the intensity value and the positive ratio value 33 (link).