β actin actb

Fresh mouse brains were collected and sliced into 2 mm sections in a mouse brain mold (RWD Company; Shanghai, China). We chose the ipsilateral cerebral hemisphere (2 mm posterior to the bregma), including the cortex and striatum, for Western blotting. Proteins were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in the protein-free rapid blocking buffer (EpiZyme; Shanghai, China) for 15 min. Then, membranes were incubated with primary antibodies against NLRP3 (1:1000; Cell Signaling Technology), ASC (1:1000; Cell Signaling Technology), CASP1 (1:1000; Santa Cruz Biotechnology), β-actin (ACTB; 1:1000; Santa Cruz Biotechnology) for 16 h at 4 °C. Next, membranes were thrice washed with tris-buffered saline containing 0.1% Tween 20 (TBST) buffer for 15 min and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) at room temperature for 1 h. The membranes were visualized using an enhanced chemiluminescence kit (ECL; Pierce; Waltham, MA, USA). Protein bands were quantified with the Image J. software.

Short‐interfering RNAs targeting EGFR#1 (5′‐CAGGAACTGGATATTCTGAAA‐3′), EGFR#2 (5′‐AAGTGTGTAACGGAATAGGTA‐3′), FASN (5′ – TGGAGCGTATCTGTGAGAA‐3′) and AKT (5′‐AATCACACCACCTGACCAAGA‐3′) were purchased from Qiagen. Scrambled (non‐targeting siRNAs, purchased from Dharmacon (D‐001810‐01), will be referred to as scrambled in the main text. Anti‐EGFR (sc‐03, 1:1,000), SREBP1 (sc‐8984, 1:1,000), FASN (sc‐20140, 1:1,000), β‐actin (ACTB, sc‐47778, 1:2,000), Lamin B (sc‐6217, 1:1,000), and α‐tubulin (sc‐8035, 1:1,000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti‐total Akt (#9272, 1:1,000), phospho‐Akt S473 (#9271, 1:1,000), NaK‐ATPase (#3010, 1:1,000), K48‐linked specific Ubiquitin (#4289, 1:1,000), Bax (#2772, 1:1,000), Bak (#3814, 1:1,000), and survivin (#2808, 1:1,000) antibodies were purchased from Cell Signaling. Anti‐tGFP antibody was purchased from Thermo Scientific (PA5‐22688, 1:1,000). Anti‐phospho EGFR Y1068 was purchased from Abcam (ab5644, 1:2,000). 2‐bromopalmitic acid (#238422), palmitate (P9767), and EGF (E9644) were purchased from Sigma. Geftinib (S1025) and Erlotinib (S1023) were purchased from SelleckChem. Xenical capsules (Orlistat) were purchased from Roche and prepared according to Knowles et al (2004), for cell culture experiments.

Cell pellets were washed twice in cold PBS, the supernatant was discarded, and cells were lysed in RIPA lysis buffer (Santa Cruz Biotechnology) containing protease inhibitors (Roche) and incubated on ice for 10 min, followed by sonication for 5 min. The sonicated samples were centrifuged for 10 min at 25° C to remove the insoluble fraction. The remaining supernatant was the whole-cell lysate. Lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Biorad). Incubations with primary antibodies recognizing ASAH1 (sc-136275), p21 (CDKN1A), p53 (TP53) (from Santa Cruz Biotechnology), as well as an antibody recognizing p16 (CDKN2A, from BD Biosciences), were followed by incubations with the appropriate secondary antibodies conjugated with horseradish peroxidase (GE Healthcare). The levels of proteins GAPDH, β-actin (ACTB), and HSP90 (from Santa Cruz Biotechnology), which displayed unchanged levels, were monitored to control for loading. Signals were developed using Enhanced Chemiluminescence (ECL) and acquired by KwikQuant Imaging System (Kindle Biosciences). Proteomic data are displayed in Supplementary Table 1.

Liver tissues were lysed with a radioimmunoprecipitation assay buffer (0.1% SDS, 150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors. Protein concentration was determined using the Bradford method. Samples (5–10 μg) were separated on 4–12% Bis-Tris gel (NuPAGE^®, Life Technologies, NP0323), and transferred to nitrocellulose membranes (Merk Millipore, HATF00010) for immunoblot analysis employing the enhanced chemiluminescence (ECL) method (Dongin LS, ECL-PS250). Antibodies against the following proteins used: ATG7 (Cell Signaling Technology, 2631, 1:1,000), FGF21 (R&D Systems, AF3057, 1:1,000), p-FRS2α (Tyr196) (Cell Signaling Technology, 3864, 1:1,000), FRS2 (Santa Cruz Biotechnology, sc-8318, 1:1,000), p-ERK (Thr202/Tyr204) (Cell Signaling Technology, 4370, 1:1,000), ERK (Cell Signaling Technology, 4695, 1:2,000), p-AKT (Ser473) (Cell Signaling Technology, 9271, 1:1,000), AKT (Cell Signaling Technology, 9272, 1:2,000), p-YAP1 (Ser127) (Cell Signaling Technology, 4911, 1:1,000), YAP1/TAZ (Cell Signaling Technology, 8418, 1:1,000), β-actin (ACTB) (Santa Cruz Biotechnology, sc-47778, 1:4,000) or HSP90 (Santa Cruz Biotechnology, sc-13119, 1:2,000). For evaluation of YAP1 dephosphorylation, densitometry of the protein bands was performed using ImageJ software (NIH).