Nupage bt gel

Control and transfected cell lines were lysed in RIPA buffer and whole protein concentration was quantified by Bradford assay (Sigma Aldrich). 20 μg of total protein extracts were separated in a 10% NuPAGE BT gel (Life Technologies) and then transferred onto nitrocellulose membranes using the iBlot system (Life Technologies). The membranes were probed with anti-hHO-1, anti-hE5NT, anti-hENTPD1 and anti-β-actin primary antibodies. For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions. Protein concentration was determined by Micro BCA Protein Assay Kit (Thermo Scientific). 10 μg of nuclear protein extracts were separated in a 4–12% gradient NuPAGE BT gel (Life Technologies) and transferred onto nitrocellulose membranes. The membranes were probed with anti-Nf-kB1 p105/p50 and anti-Lamin B2 primary antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence (SuperSignal West Dura, Thermo Scientific) and digitally acquired using G:BOX (Syngene) instrument.
Densitometric analysis on Nf-kB/p50 and Lamin B2 bands was performed by using “Gel analyzer” function of ImageJ software[34 (link)].