Western blotting

Maternal pancreas tissues obtained from second pregnancies were homogenized in RIPA lysis buffer (Santa Cruz Biotechnology; catalog No. sc-24948A) supplemented with Halt Protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog No. 78443). Protein concentrations were determined by the DC protein assay (Bio Rad). A total of 50 μg of protein per reaction sample were separated on 4–20% ExpressPlus PAGE Gels (GenScript, Piscataway, NJ, USA; catalog Nos. M42012 and M42015) and transferred to PVDF Blotting Membrane (GE Healthcare; catalog No. 10600023). Following transfer, membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20, for non-specific binding and subsequently probed with specific primary antibodies to SRY-box 17 (SOX17; 1:500, Santa Cruz Biotechnology, catalog No. sc-130295), phosphorylated signal transducer and activator of transcription 5 (STAT5^pTyr694, DH47E7, 1:500, Cell Signaling Technology, catalog No. 4322), PDX1 (D59H3; 1:1000, Cell Signaling Technology, catalog No. 5679) and glyeraldehyde-3-phosphate dehydrogenase (GAPDH, 1:300, Abcam, catalog No. ab9485). Immunoreactive proteins were visualized by Luminata Crescendo Western HRP Substrate (Millipore, catalog No. WBLUR0500) according to the manufacturer’s protocol.