Western ecl substrate mixture

Protein was extracted from liver tissue or cell cultures as described [25 (link)]. The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (ThermoFisher Scientific). Proteins (30 μg/sample) were subjected to 12% SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Monoclonal anti-rabbit PTEN (9188), Akt (4691), p-Akt (Ser473) (4060), GSK3β (12456), p-GSK3β (Ser9) (5558), NICD (3608), NRF2 (12721), STING (13647), p-STING (Ser366) (50907), TBK1 (38066), p-TBK1 (Ser172) (5483), IRF3 (11904), p-IRF3 (Ser396) (29047), P65 (8242), p-P65 (Ser356) (3033), RIPK3 (10188), p-MLKL(Ser345) (37333), Lamin B2 (24209), β-actin Abs (12262) (CST), p-Presenilin 1/PS-1 (Ser357) (ab78914), Presenilin 1/PS-1 (ab76083) and RBPjκ (ab25949) (Abcam) were used to overnight incubation at 4℃. Then add a secondary antibody solution and incubate for 1 h. Rinse the blot 5–7 times with TBST. The Western ECL substrate mixture (Bio-Rad) was used to image with the iBright FL1000 (ThermoFisher Scientific).

Protein was extracted from liver tissue or cell cultures as described [7 (link)]. Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). The buffer contains 1% proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). The Foxo1, p-JNK, JNK, p-Akt, p-β-catenin, β-catenin, Snail, HMGB1, RIPK3, p-MLKL, NLRP3, cleaved caspase-1, Lamin B2, β-actin (Cell Signaling Technology), Gli1 (Santa Cruz Biotechnology), Sonic Hedgehog (Shh), SMO, NEK7, and TCF4 (Abcam) mAbs were used. The membranes were incubated with Abs, and then added Western ECL substrate mixture (Bio-Rad) for imaging with the iBright FL1000 (ThermoFisher Scientific). Relative quantities of protein were determined by comparing to the β-actin expression, using iBright image analysis software (ThermoFisher Scientific).