Proteins from frozen pNEN tissue specimen (60–80 mg) pulverized in liquid nitrogen or from BON1 cells pellets were extracted using RIPA buffer containing complete protease inhibitors (Sigma-Aldrich) and supplemented with phosphatase inhibitor sets 1 and 2 (Sigma-Aldrich). Using an ultrasonic homogenizer (SonoPuls mini20 Bandelin®, Berlin, Germany), the suspensions were subjected to a 30-s sonication step on ice (ampl. 80%, 0.99 kJ) and subsequently centrifuged at 16,000 g for 10 min.
Supernatants were collected and divided into aliquots, and the total protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions.
Western blot was performed as previously described (37 (link)) using the following monoclonal antibodies: anti-CRP, anti-IL-6, anti-total-STAT3 (all Abcam), anti-phospho-STAT3 (Tyr705), anti-total-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-total-AKT and anti-phosho-AKT (Ser473) (all Cell Signaling). The secondary antibody used was goat anti-rabbit (R&D Systems). Quantification of protein expression was performed using ImageJ (EHD imaging, Damme, Germany).
Supernatants were collected and divided into aliquots, and the total protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions.
Western blot was performed as previously described (37 (link)) using the following monoclonal antibodies: anti-CRP, anti-IL-6, anti-total-STAT3 (all Abcam), anti-phospho-STAT3 (Tyr705), anti-total-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-total-AKT and anti-phosho-AKT (Ser473) (all Cell Signaling). The secondary antibody used was goat anti-rabbit (R&D Systems). Quantification of protein expression was performed using ImageJ (EHD imaging, Damme, Germany).