Peroxidase labeled secondary antibody

A total of 3 × 10⁵ NPCs were seeded on 6 well Matrigel-coated plates and differentiated to neurons. Soluble cellular extracts for immunoblotting were obtained by lysing cells in 20 mM Tris–HCl, pH 7.4, 1% Triton X-100, and a protease inhibitor cocktail (Roche, Monza, Italy) followed by centrifugation at 16,000 g for 10 min. Soluble proteins (25 µg) were separated by sodium dodecyl sulfate–12% poly-acrylamide gel electrophoresis (SDS-PAGE), and immunoblotting was performed using specific antibodies: mouse anti spectrin α-chain (1:1000; MAB1622, Merck-Millipore, Milano, Italy); mouse anti calpain1 (1:1000; MA1-1679, ThermoFisher Scientific); mouse anti β-actin (1:6000; A5441, Sigma-Aldrich) followed by peroxidase-labeled secondary antibodies (Sigma-Aldrich). The signal was then revealed using the ECL-chemiluminescence kit (GE Healthcare, Milano, Italy) and detected with ChemiDoc MP Imaging System (BIORAD, Segrate, Italy). Total protein contents were measured using the BCA protein assay calibrated with bovine serum albumin (ThermoFisher Scientific).

To determine the expression level of the fusion proteins, SDS-PAGE and Western Blot analysis of cytoplasmic cell lysates was performed. Cell extracts were prepared in lysis buffer (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 10% (v/v) glycerol, 1 mM Na₃VO₄, 10 mM Na₄P₂O₇, 10 mM NaF, 1 mM EDTA, 10 µg/mL leupeptin, 1 mM PMSF, and 1% (v/v) Triton X-100) and protein content was determined by BCA assay (Thermo Scientific, Carlsbad, CA, USA). After SDS-PAGE electrophoresis, proteins were transferred to nitrocellulose membranes. Proteins were immunodetected by using appropriate primary antibodies and peroxidase-labeled secondary antibodies (Sigma–Aldrich, St Louis, MO, USA). Bands were visualized with Pierce ECL or ECL Plus Western Blotting Substrate (Thermo Scientific). Specific antibodies against the following proteins were used as follows: Bcl-X_L (Cell Signaling Technology, Danvers, MA, USA); Bcl-2 (Abcam, Cambridge, UK); Mcl-1 (Santa Cruz Biotechnology, Dallas, TX, USA); Bim (Merck Millipore, Darmstadt, Germany); Puma (Abcam); Noxa (Santa Cruz Biotechnology); Bax (BD Biosciences); and Bak (Santa Cruz Biotechnology and Millipore, Darmstadt, Germany). The protein-loading control was achieved by membrane reprobing with an anti-β-actin or anti-α-tubulin antibody (Sigma–Aldrich).

Immunoblotting was performed as described earlier [48 (link)]. Briefly, cellular lysates were prepared by harvesting cells in Laemmli sample buffer. Proteins were separated on SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. After blocking, the membranes were incubated with specific primary antibodies overnight, washed and then incubated with peroxidase-conjugated secondary antibodies. Finally, peroxidase activity was detected with ECL+ reagents (AP Biotech, Prague, Czech Republic) using a LAS-4000 CCD camera (AP Biotech, Prague, Czech Republic). Specific antibodies against PARP were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) peroxidase-labeled secondary antibodies was from Sigma Aldrich (Prague, Czech Republic, peroxidase-labeled secondary antibodies) or were generously gifted by Dr. B. Vojtěšek (p53, p21WAF1, PCNA).