Anti total stat3

Proteins from tissue (1 × 2 mm) or BON1 cells were extracted using RIPA buffer containing complete protease inhibitors (Sigma-Aldrich) according to the manufacturer’s instructions, and the protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions. Western blots were processed as previously described (31 (link)) using the following antibodies: anti-IL-6, anti-total-STAT3, and anti-ATX (all from Abcam); and anti-phospho-STAT3 (Tyr705) (Cell Signaling Technology). A goat anti-rabbit secondary antibody was used (R&D Systems). Protein expression was quantified using ImageJ (NIH).

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).