Ab198921

After the extraction of proteins from serums and cells via radioimmunoprecipitation assay (RIPA) lysis solution (Beyotime, Shanghai, China), 60 μg quantified proteins were isolated on 10% sodium dodecyl sulfate–polyacrylamide gel for 2 h, followed by the transferring of proteins onto the polyvinylidene fluoride membranes (Beyotime). Then, 5% skim milk (Beyotime) was applied for blocking membranes for 3 h, which were incubated with primary antibodies from Abcam (Cambridge, United Kingdom): anti-Hexokinase 2 (anti-HK2; ab209847, 1:1000), anti-Glucose Transporter 1 (anti-GLUT1; ab115730, 1:1000), anti-SOX13 (ab198921, 1:1000) and anti-GAPDH (ab9485, 1:3000) overnight at 4 °C Following the incubation of secondary antibody (Abcam, ab205718, 1:5000) for 1 h, the immunoreactive signals were assayed through enhanced chemiluminescence reagent (Abcam), and the gray levels were analyzed via the Image J software (NIH, Bethesda, MD, USA).

4-µm Formalin-fixed paraffin-embedded pancreatic tumour sections of tissue blocks were cut using a microtome, mounted onto poly-l-lysine-coated slides, and dried overnight at 37°C. Slides were stored at room temperature until required. Briefly, the slides were immunohistochemically stained using the primary antibody specific for SOX13 (Abcam ab198921). After deparaffinization and rehydration, the slides were subjected to an antigen retrieval step consisting of 20-min incubation in pH 9.0 buffer (Target Retrieval Dako) in a 95°C water bath followed by cooling to room temperature. Staining was performed using an automated staining apparatus for IHC (Autostainer; Dako) according to the manufacturer's guidelines. Positive and negative controls were processed at the same time. The slides were counterstained with haematoxylin, dehydrated in graded alcohols, and mounted (DPX; Sigma). Scoring and interpretation were determined based on the intensity of staining in tumour using a semiquantitative scoring system. The intensity score was assigned as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) by a pathologist (MC).