Phenylmethanesulfonyl fluoride

The cells were lysed in RIPA lysis buffer (DingGuo Biotechnology, Co., Ltd., Beijing, China) supplemented with phenylmethanesulfonyl fluoride (DingGuo Biotechnology, Co., Ltd.) and centrifuged at 10,000 × g for 5 min at 4°C. The protein samples were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the products were electrotransferred to nitrocellulose filter membrane (GE Healthcare Life Scientific, Chalfont, UK). Rabbit anti-SYK polyclonal antibody (cat. no. sc-1077; dilution, 1:700; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-GAPDH monoclonal antibody (cat. no. 60004-1-lg; dilution, 1:4,000; Proteintech, Rosemont, IL, USA) were used for immunodetection. Following incubations with primary antibodies at 4°C overnight, IRDye^® 800CW-conjugated goat anti-rabbit and IRDye^® 680RD-conjugated goat anti-mouse secondary antibodies (cat. nos. P/N 925-32211 and P/N 925-68070, respectively; dilution, 1:10,000; LI-COR Biosciences, Lincoln, NE, USA) were incubated with the membranes for 1 h at room temperature. Proteins were detected using the Odyssey^® CLx Imaging System (LI-COR Biosciences), and the band intensities were analyzed using the Odyssey Application 3.0 software (LI-COR Biosciences).

COS7 cells were grown in T25 flasks to 70% confluence, and then transfected with pcDNA3.1(+)-S100A6-HA individually or together with pcDNA3.1(+)-SAG1-3×Flag for 48 hrs. The cells were washed for 3 times with PBS and lysed with cell lysis buffer (Beyotime, China) containing 1 mM phenylmethanesulfonyl fluoride (Dingguo Changsheng Biotechnology, China). Cell lysates were centrifuged at 14,000×g, 4°C, for 10 min. The supernatants were collected and incubated with Anti-FLAG® M2 antibody (Sigma Aldrich, USA) for 2 hrs at 4°C with gentle rotation. Then, 20 μL protein A-agarose (Santa Cruz Biotechnology, USA) was added to the mix and incubated overnight at 4°C with gentle rotation. The beads were collected by centrifugation at 500×g for 5 min at 4°C and then resuspended in 6×SDS-PAGE sample buffer. The samples were boiled, loaded onto the gels for SDS PAGE and then analyzed by WB.

Following treatment with different concentrations (40, 50 and 60 µM) of 20(S)-PPD, protein from HepG2 cells was extracted by a radioimmunoprecipitation assay buffer containing 1% phenylmethane sulfonyl fluoride (both Beijing Dingguo Changsheng Biotechnology Co., Ltd.), then proteins were determined using a BCA protein assay kit. Proteins (20 µg/lane) were then loaded onto a 12% polyacrylamide-SDS gel. The gel was subsequently blotted onto a PVDF membrane and blocked with 5% (w/v) non-fat milk for 1 h at room temperature. The membrane was then incubated with cleaved caspase-3 (cat. no. 9664S), cleaved caspase-9 (cat. no. 7237T), PARP (cat. no. 9532T), Bcl-2 (cat. no. 4223T), Bax (cat. no. 5023T), Akt and p-Akt (Ser 473) were from the Phospho-Akt Pathway Antibody Sampler kit (cat. no. 9916T) and β-actin (cat. no. TA-09) primary antibodies at 1:1,000 dilutions at 4°C overnight. Primary antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase The goat-anti-mouse (cat. no. IH-0031) and goat-anti-rabbit (cat. no. IH-0011) secondary antibodies were used at 1:5,000 dilution at room temperature for 1 h, and visualized using a BeyoECL Plus enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) ImageJ software (version 1.5.0.26; National Institutes of Health, Bethesda, MD, USA) was used for analysis.