Western lightning chemiluminescense reagent plus kit

Bisphenol A [2,2-bis (4-hydroxyphenyl) propane] (BPA) (239658) and nonylphenol (NP) (442873) were obtained from Sigma (St. Louis, MO, USA). LipofectAMINE 2000 (11668-027), p-nitrophenyl phosphate (p-NPP) (002212), substrate of alkaline phosphatase, TRIzol-Reagent (15596026), FURA2-AM (F1221), and propidium iodide (P1304MP) were obtained from Invitrogen (Carlsbad, CA, USA). BB-94 (Batimastat) (196440) was obtained from Calbiochem (San Diego, CA, USA). ADAM17 antibody (ab39163) was purchased from Abcam (Cambridge, MA, USA). PARP-1/2 antibody (sc-7150) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cleaved Caspase-3 (Asp175) antibody (#9661) was acquired from Cell Signaling (Danvers, MA, USA). β-actin (AC-15) and anti-rabbit IgG-FITC (F0382) antibodies were purchased from Sigma (St. Louis, MO, USA). Peroxidase anti-mouse IgG (5220-0286) and peroxidase anti-rabbit IgG (5220-0283) antibodies were obtained from KPL (Gaithersburg, MD, USA). Human phospho-kinase antibody array (Catalog #ARY003B) was obtained from R&D Systems (Minneapolis, MN, USA). Western Lightning Chemiluminescense Reagent Plus kit (NEL104001EA) was obtained from PerkinElmer Inc. (Waltham, MA, USA).

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).