Imagequant las 4000 chemiluminescence and fluorescence imaging system

Cells were lysed in 20 mM Tris‐HCl (pH 7.5) containing 1% NP40, 0.2% Triton X‐100, 150 mM NaCl, 2 mM EDTA, 1.5 mM MgCl₂ with phosphatase and protease inhibitors. Lysates were centrifuged at 16,000 × g and 4°C for 10 min to remove cell debris and nuclei. The resulting supernatants were diluted in Laemmli sample buffer with or without 2‐mercaptoethanol (0.15 M), and separated electrophoretically in corresponding poly‐acrylamide SDS‐PAGE. Upon transfer to nitrocellulose membranes, the extracts were subjected to western blot analyses. To this end, the filters were blocked with 5% BSA in TBS‐Tween 0.1%, incubated overnight with the primary antibodies, and the immunoreactive bands visualized upon incubation with peroxidase‐labelled secondary antibodies for 30 min. Signal detection was performed using the ImageQuant LAS‐4000 chemiluminescence and fluorescence imaging system (Fujifilm) (Blas‐Rus et al., 2017 (link)).

Whole cell lysates were obtained by direct lysis of the cells using an ice-cold Mammalian Protein Extraction Reagent (M-PER, Pierce). Nuclear and cytoplasmic fractionations were performed using the Nuclear and Cytoplasmic Extraction Kit (Pierce). Protein (20 μg) was resolved by 10 % SDS-PAGE and electro-transferred onto a polyvinylidene difluoride membrane. Western blotting was performed according to standard methods, using anti-cleaved-PARP, anti-p53, anti-cytochrome c, anti-Akt, anti-phosphorylated-Akt (ser473), anti-FOXO3a, anti-p21, anti-p27 and anti-β-actin antibodies (Cell Signaling Technology). The membranes were developed using an enhanced chemiluminescence detection system, horseradish peroxidase substrate (Millipore) and an ImageQuant LAS-4000 Chemiluminescence and Fluorescence Imaging System (FujiFilm).