Horseradish peroxidase labeled secondary antibody

Human hepatoma cell line SMMC7721 was purchased from the Cell Bank at the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Lentivirus vector expressing AQP9-enhanced green fluorescent protein (eGFP) (named LV-AQP9) and lentivirus vector expressing eGFP (named LV-PWPI) were obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit was purchased from Roche (Basel, Switzerland). Cell counting kit-8 (CCK8) assay was purchased from Dojindo (Tokyo, Japan). Rabbit anti-human AQP9 antibody was purchased from Abcam (Cambridge, MA, USA). Rabbit anti-human caspase-3, PI3K, proliferating cell nuclear antigen (PCNA), Akt, P-Akt, and forkhead box protein O1 (FOXO1) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody and horseradish peroxidase-labeled secondary antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).

The total protein was extracted from 60 mg lung tissue using RIPA lysis buffer. Concentration of the protein was quantified by quantitative bromochloroacetic acid (BCA) protein kit (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). The protein was mixed with 4 × SDS-PAGE loading buffer (dilution rate 3 : 1) and boiled at 100°C for 10 min to make it denatured. Equal amounts of protein (30 μg per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into 0.45 μm polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat-dried milk at room temperature for 2 h, membranes were incubated with primary antibodies overnight at 4°C. Membranes were detected with relevant horseradish peroxidase-labeled secondary antibodies (Zhong Shan Golden Bridge Biotechnology Co., Ltd., Beijing, China; 1 : 20000) for 1 h. The protein band was visualized by an electrochemiluminescent (ECL) reagent and exposed to X-film. GAPDH or β-actin was used for normalization. The mean density of each protein band was measured by ImageJ software (National Institutes of Health, USA). The description and concentration of primary antibodies used in WB analysis are listed in Table 2.