Cold lysis buffer

Islet pellets were dislodged into cold lysis buffer from Cell Signaling Technologies (CST, USA) supplemented with protease inhibitor cocktail tablets (Roche Applied Science, Basel, Switzerland). The homogenized tissue was sonicated and whole protein extracts were recovered by cold centrifugation (12000× g for 30 min). Protein extracts (50 µg) were incubated for 3 hours at 4°C with a c-Jun (1–79)-GST fusion protein coupled to glutathione beads (1 µg, Sigma Aldrich, Switzerland). After a short centrifugation (900× g for 3 min), the supernatants were discarded, and pellets were washed twice, then mixed to kinase buffer (20 mmol/l HEPES pH 7.5, 20 mmol/l β-glycero-phosphate, 10 mmol/l MgCl₂, and 1 mmol/l DTT) and incubated (30 min at 30°C) with 0.5 µl ^[γ-33]ATP (111 TBq/mmol, PerkinElmer, Switzerland). The reactions were terminated by addition of Laemmli sample buffer (50 mmol/l Tris-HCl pH 6.8, 2% [w./vol.] SDS, 100 mmol/l DTT, 0.1% [w./vol.] bromophenol blue and 10% [vol./vol.] glycerol. GST-c-Jun phosphorylation was resolved by SDS-PAGE, (BIO-RAD, Switzerland) and then, gels were fixed, dried and exposed overnight to X-ray film. The protein band intensities were quantified by densitometric analysis using the ImageJ processing software.

For Western blot analysis, 5 x 10⁶ Ba/F3 cells were washed three times in PBS, left 60 minutes with only RPMI-1640 medium supplemented with 10% FCS and then incubated with 100 ng/mL NRG1 (Peprotech) for 30 minutes. Total cell lysates were obtained by lyzing cells in cold lysis buffer (Cell Signaling Technology) supplemented with complete protease inhibitor tablets (Roche). Thirty microgram of protein lysate was combined with SDS loading buffer plus DTT (Cell Signaling Technology) before electrophoresis on 4–12% NUPAGE gels (Invitrogen) and the proteins were transferred to nitrocellulose membranes. Antibodies used were as follows: anti-ERBB3 (sc-285; Santa Cruz Biotechnology), anti-phospho-ERBB3 (AF5817; R&D Systems) anti-ERBB4 (#4795; Cell Signaling Technology), anti-phospho-ERBB4 (#4757; Cell Signaling Technology), anti-Akt (#9272; Cell Signaling Technology), anti-phospho-Akt (#4060S; Cell Signaling Technology), anti-MEK (#9126; Cell Signaling Technology), anti-phospho-MEK (#9154; Cell Signaling Technology) and anti-rabbit peroxidase-labeled antibodies (AP Biotech, Uppsala, Sweden). The LAS-3000 Imaging System (Fujifilm Global) was used for detection.