Protein Extraction and Western Blotting Protocol

Protein extraction and Western blotting were performed as previously published [23 (link)]. Briefly, the homogenization of cells was performed in a buffer containing 1M NaCl, 1mM EDTA, 10 mg/mL PMSF, 1% Triton X-100, and 20mM Tris–HCl pH 7.4, plus a general metalloprotease inhibitor, BB-94 10 µM, and protease inhibitor cocktail (Sigma), and then centrifuged for 10 min at 10,000× g at 4 °C. Then, samples were run on a 10% polyacrylamide gel under reducing and denaturing conditions, transferred to nitrocellulose at 400 mA for 1 h, and blocked with 3% (w/v) non-fat milk, 0.1% Tween in TBS, pH 7.4. Primary antibodies were incubated overnight at 4 °C with the following dilutions: anti-ADAM17 and anti-PARP-1/2 (0.2 µg/mL), or anti-β-actin (0.3 µg/mL). A secondary antibody conjugated with horseradish peroxidase (0.3 µg/mL) (KPL, Gaithersburg, MD, USA) was used. Protein bands were developed using the Western Lightning Chemiluminescense Reagent Plus kit (PerkinElmer Inc, Waltham, MA, USA).