Ecl detection reagents

Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1 mM EDTA) together with a complete (EDTA-free) protease inhibitor cocktail (Kodak, Rochester, NY, USA), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were resolved by SDS-PAGE, transferred to Hybond enhanced chemiluminescence (ECL) Nitrocellulose membranes (Amersham Biosciences, Freiburg, Germany), immunoprobed with antibodies, and visualized by ECL detection reagents (Kodak, Rochester, NY, USA). The primary antibodies of ARPP-19 (sc-135145), ERRγ (sc-133561), Cyclin D1 (sc-753) and c-Myc (#9402) were purchased from Santa Cruz Biotechnology or Cell Signaling. As described in Liu et al.29 (link)
and Yin M et al.14 (link), protein levels were normalized to GAPDH and quantified using Tanon Gel image system (Tanon, Shanghai, China).

Immunoblot assay was performed as previously reported (Liao et al., 2021a (link)). In short, pan proteins were extracted from HCC cells using RIPA buffer supplemented with phosphatase inhibitor cocktail on the ice. Cell lysates were then sonicated for three times (10 s/time) to break the DNA. Protein determination was performed using BCA assay. After determination and denaturation, 20 μg proteins were loaded onto and disassociated by 12% SDS–PAGE gels, followed by the transference to polyvinylidene difluoride membranes and blockade with non-fat 5% milk for 1 h. Then, the membranes were washed with PBS-T for three times and incubated with diverse primary antibodies at 4°C overnight. The working concentration of primary antibodies were 1:1,000. After the reaction and wash with PBS-T, all membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Finally, the membranes were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan). Each membrane was stripped less than thrice.

Cell lysates were prepared by treating cells with ice-cold lysis buffer (20 mM Tris pH 8.0, 1% NP40, 10% glycerol, 137 mM NaCl, 10 mM EDTA pH 8.0, and Roche Applied Science (Roche Applied Science, Monza, Italy) protease inhibitor cocktail “Complete”) for 20 min, followed by high-speed centrifugation at 4 °C for 15 min [59 (link),60 (link)]. Proteins (50 µg) were separated on 12% SDS-PAGE gels and then transferred to polyvinylidene difluoride membrane.
The blots were blocked with 5% non-fat dry milk in 20 mM Tris/HCl, pH 7.5, 500 mM NaCl plus 0.1% Tween 20 (TBS-T). The membranes were subsequently incubated in agitation at 4 °C overnight in 1% BSA-TBS-T buffer containing the specific antibody against: p-ERK (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Erk 1/2 (cell Signaling cat n°9102 1:1000 3%BSA), EGFR (Santacruz sc-03 1:500 3%Milk O.N.) and tubulin (Santacruz sc-5286 1:1000 3%milk O.N.).
After washing four times with TBS-T, the blots were incubated for 1 h at room temperature (RT) with the second antibody conjugated to peroxidase, washed four times with TBS-T, developed with ECL detection reagents (Amersham, Little Chalfort, Buckinghamshire, UK) for 1 min and exposed to X-Omat film (Eastman Kodak Co., Rochester, NY, USA). The whole western blot figures can be found in the supplementary figures.