Anti acc1

Total protein was extracted from the cells using RIPA buffer (Boster, China) containing a phosphatase inhibitor and quantified using the bicinchoninic acid protein detection kit (Invitrogen, USA) according to the manufacturer’s instructions. After denaturation, 20 mg of protein samples were loaded into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel for electrophoresis. After transfer of protein bands, the polyvinylidene fluoride membrane (Invitrogen, USA) was blocked with bovine serum albumin (Invitrogen, USA) at 25°C for 1 h and incubated with the following primary antibodies overnight at 4°C: anti-phospho-EGFR (1:1,000, Abclone), anti-phosphorylated PI3K (1:1,000, Abclone), anti-phosphorylated AKT (1:1,000, Abclone), anti-phosphorylated NF-κB (1:1,000, Abclone), anti-SREBP1 (1:1,000, Abcam), anti-ACC1 (1:1000, Proteintech), anti-FASN (1:1,000, Proteintech), and β-actin (1:1,000, Abclone). After washing with Tris-buffered saline with 0.1% Tween^® 20 (TBS-T), the membrane was incubated with an anti-rabbit secondary antibody for 2 h at room temperature. After another washing with TBS-T, protein bands were visualized using an enhanced chemiluminescent substrate kit (Obsen, Beijing). Experiments were performed in triplicate.

To obtain total protein lysates, triturated tissue or cell pellets were lysed with cell lysate‐containing mixed protease inhibitors. The lysate was incubated on ice for 30 min and centrifuged at 12,000 g for 15 min at 4°C. The protein concentration of each sample was determined using the bicinchoninic acid (BCA) assay. Samples were loaded on 10% SDS‐PAGE. After electrophoresis, proteins in the gel were transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk in TBST for 2 h at room temperature, then incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 2 h. The main antibodies include anti‐ACLY (Proteintech, Wuhan, China), anti‐SREBF1 (Proteintech, Wuhan, China), anti‐MOGAT2 (Proteintech, Wuhan, China), anti‐ACC1 (Proteintech, Wuhan, China), anti‐ACS (Proteintech, Wuhan, China), anti‐FASN (Proteintech, Wuhan, China), anti‐β‐actin (Proteintech, Wuhan, China), pan‐acetylated antibody (AC) (PTMBIO, Hangzhou, China), anti‐IgG (Proteintech, Wuhan, China), anti‐SIRT2 (Proteintech, Wuhan, China), anti‐HDAC1 (Proteintech, Wuhan, China) and anti‐PCAF (Proteintech, Wuhan, China). Immunoreactive bands were observed with an Azure C300 system.

Freshly collected tissue samples were obtained from the 32 SACC patients and the 32 patients of benign salivary gland tumor underwent surgery, who were registered between November 2016 and October 2020 in the Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of Qingdao University. The experimental protocols were approved by the Institutional Ethics Committee of the Affiliated Hospital of Qingdao University. Every patient was understanding and signed separate informed consent forms for sampling and molecular analysis. The study methodologies conformed to the standards set by the Declaration of Helsinki. None of the SACC patients had undergone preoperative radiotherapy or chemotherapy. The tissue samples were fixed in 10% formaldehyde and embedded in paraffin. Sections were prepared, and immunohistochemical staining was conducted using a conventional protocol. Briefly, sections were incubated with anti‐PRRX1 (1 : 80; Novus Biologicals, Centennial, CO, USA), anti‐ACC1 (1 : 500; Proteintech, Rosemont, IL, USA), and anti‐Beclin‐1 (1 : 500; Proteintech) for 2 h, followed by incubation with secondary antibody. Then, colorimetric detection was performed using a DAB kit (ZSGB BIO).