Total protein extraction was performed as previously described in ref. 27 (link).
Nuclear and cytoplasmic proteins fractionation, was performed using buffers A and B (A: Hepes 10 mM, KCl 10 mM, EDTA 0,2 mM, DTT 1 mM, PMSF 1 mM, okadaic acids 1 μM, cocktail of phosphatase and protease inhibitors; B: Hepes 20 mM, NaCl 0,4 M, EDTA 0,2 M, DTT 1 mM, PMSF 1 mM, okadaic acids 1 μM, cocktail of phosphatase and protease inhibitors and glicerol 10% (v/v)).
Western blot (WB) was performed as previously described in ref. 27 (link). The following primary antibodies were used: PARP (#9542), LC3B (2775 S), phospho FOXO3a Ser318-321 (9465 S), phospho p65 Ser536 (#3031), CK1α (2655 S) (Cell Signaling Technology, MA, USA); β-actina (A5441) (Sigma-Aldrich, Italy); GAPDH (MAB374) (Millipore, USA); p62 (#P0067), FOXO3a (ab12162), p65 (ab7970-1) (ABCAM, UK). Anti-rabbit-HRP (Cell Signaling, USA) and anti-mouse-HRP (KPL, USA) were used as secondary antibodies. Acquisition of the bands was performed in chemiluminescence using the Image Quant LAS 500 machine (GE Healthcare, USA) and the densitometric analysis of the bands was performed with the Image Quant TL software (GE Healthcare, USA).
Nuclear and cytoplasmic proteins fractionation, was performed using buffers A and B (A: Hepes 10 mM, KCl 10 mM, EDTA 0,2 mM, DTT 1 mM, PMSF 1 mM, okadaic acids 1 μM, cocktail of phosphatase and protease inhibitors; B: Hepes 20 mM, NaCl 0,4 M, EDTA 0,2 M, DTT 1 mM, PMSF 1 mM, okadaic acids 1 μM, cocktail of phosphatase and protease inhibitors and glicerol 10% (v/v)).
Western blot (WB) was performed as previously described in ref. 27 (link). The following primary antibodies were used: PARP (#9542), LC3B (2775 S), phospho FOXO3a Ser318-321 (9465 S), phospho p65 Ser536 (#3031), CK1α (2655 S) (Cell Signaling Technology, MA, USA); β-actina (A5441) (Sigma-Aldrich, Italy); GAPDH (MAB374) (Millipore, USA); p62 (#P0067), FOXO3a (ab12162), p65 (ab7970-1) (ABCAM, UK). Anti-rabbit-HRP (Cell Signaling, USA) and anti-mouse-HRP (KPL, USA) were used as secondary antibodies. Acquisition of the bands was performed in chemiluminescence using the Image Quant LAS 500 machine (GE Healthcare, USA) and the densitometric analysis of the bands was performed with the Image Quant TL software (GE Healthcare, USA).