Prss8

Tissue or cell lysates were prepared by homogenization in T-PER or M-PER (Thermo Fisher Scientific). Protein lysates were subjected to SDS–polyacrylamide gel electrophoresis and probed with the following primary antibodies: PRSS8 (BD Biosciences), EGF (Santa Cruz), EGFR (Cell Signaling Technology), p-EGFR (Tyr1068, Cell Signaling Technology), Akt-1 (Santa Cruz), p-Akt (Ser473, Cell Signaling Technology), Erk1/2 (p44/42 MAPK, Cell Signaling Technology), p-Erk1/2 (Cell Signaling Technology), β-actin (Cell Signaling Technology), and Na + /K + -ATPase α (Santa Cruz). For the detection of EGFR and p-EGFR, 3%–8% Tris–acetate gels (Thermo Fisher Scientific) were used, and for others, 4%–20% Tris–Glycine gels (Thermo Fisher Scientific) were used. At least three technical replicates were performed for all blots. Full-length blots are presented in the Supplementary files.

Mice were deeply anesthetized and perfused through the heart with 4% paraformaldehyde (PFA) phosphate buffer (PB) solution (FUJIFILM Wako). The samples were fixed in 4% PFA and subsequently embedded in paraffin. Then, 3-mm sections were prepared and stained with hematoxylin and eosin, whereas other sections were processed for immunohistochemistry. Nonspecific binding of the antibodies was blocked with 3% BSA before incubation overnight at 4 °C with the primary antibodies. The following primary antibodies were used for immunostaining: PRSS8 (BD Biosciences and PA5-27,977, Thermo Fisher Scientific), EGFR (Cell Signaling Technology), p-EGFR (Cell Signaling Technology), and insulin (Dako). Secondary antibodies included Alexa-conjugated goat antimouse, antirabbit, and antiguinea pig immunoglobulin G (IgG) antibodies.