Specific horseradish peroxidase conjugated secondary antibody

To assess expression of B7H6 protein, we first homogenized whole cells or patients’ pancreatic tissue in 10 volumes of RIPA lysis buffer containing (in mM) 1 PMSF, 1 DTT, and 0.02% (v/v) protease cocktail (Sigma Aldrich, St. Louis, MO, USA). The homogenates were centrifuged at 20,000×g at 4°C for 15 min, and the supernatants were retained. Protein concentrations were determined by the BCA method. Equal amounts of protein from each sample were loaded onto SDS–PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 3% BSA/TBS-T for 1 hour at room temperature and then incubated with B7H6 antibody (MAB7144, Minneapolis) or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (ab9484, Abcam) in 1% BSA/TBS-T overnight at 4 °C. Following washing, the nitrocellulose membranes were incubated with a specific horseradish peroxidase-conjugated secondary antibody (DAKO, 1:5000) for 1 hour at room temperature, washed with PBS, and visualized with ECL (Thermo Fisher). Signals were detected with the FluorChem E system (Protein Simple, San Jose, CA). Each band was normalized to an internal control and quantified with ImageJ software.

Cells were lysed in RIPA lysis buffer. Proteins were separated by SDS–PAGE and transferred on a nitrocellulose membrane. The membrane was blocked with 3% BSA/PBS for 1 h at RT followed by incubation with the corresponding primary antibody in 1% BSA/PBS-T overnight at 4 °C: pTyr (Millipore, #05-321, 1:500), Tie2 (R&D, #AF313, 1:1,000), Tie2 (R&D, #AF762), pAKT (Cell Signaling, #4060S, 1:1,000), AKT (Cell Signaling, #9272S, 1:1,000), FOXO1 (Cell Signaling, #9454, 1:1,000), pFOXO3A (Cell Signaling, #9466, 1:1,000), FOXO3A (Cell Signaling, #12829, 1:1,000), VEGFR2 (Cell Signaling, #2479, 1:1,000), tubulin (Sigma, #T8203, 1:5,000). Following washings, membranes were incubated with a specific horseradish peroxidase-conjugated secondary antibody (DAKO, 1:5,000) for 1 h at RT, washed and proteins were visualized by incubation with ECL (Thermo Fisher). Quantification was performed in Fiji. Uncropped western blots/arrays and larger blot/array areas are presented in Supplementary Figs 15–19.