Tanon imaging system

Both fresh tissues and cultured cells were homogenized and lysed in RIPA buffer containing protease inhibitors. The supernatant was collected after centrifuging at 13,000 g for 15 min at 4 °C, and total protein concentration was determined with a BCA protein concentration assay kit (Beyotime, Shanghai, China). Equal quantities of obtained protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat-dried milk dissolved in TBST buffer (TBS containing 0.1% Tween-20), the membranes were further incubated with primary antibodies overnight at 4 °C. Then membranes were incubated with secondary antibodies at room temperature for 1 h. Immunoreactive bands were visualized using a Tanon imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China).

After treated with 4 μmol/l of rPK5-RL-Gal-3C for 48 h, HCC cells were harvested and lysed in RIPA buffer with protease inhibitor on ice for 30 min. Then the supernatant was collected after centrifuging at 13, 000 g for 15 min and the protein concentrations were measured by using BCA protein concentration assay kit (Vazyme, Nanjing, China). Subsequently, 30 μg of protein was separated with SDS-PAGE and transferred to the PVDF membrane. After blocked with 5% nonfat-dried milk for 1 h at room temperature, the membranes were cropped according to the related protein molecular weight. Only targeted blots were reserved and incubated with primary antibodies overnight at 4 °C. Following incubated with secondary antibodies for 1 h at room temperature, the immunodetection of the membrane was performed using Tanon imaging system (Tanon Science & Technology Co, Ltd, Shanghai, China).

At the end of reperfusion or reoxygenation, heart tissue or cell culture was collected, and proteins were isolated using radio-immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Roche), and subsequently quantitated by BCA kit (Pierce Chemical Company, Rockford, IL, United States). Equal amount of proteins were loaded and separated by 10 or 12% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h, and subsequently incubated with primary antibodies against p-ERK (1:1000; Cell Signaling Technology/CST, Danvers, MA, United States), ERK (1:1000; Proteintech, Wuhan, China), p-Akt (1:1000; CST), Akt (1:1000; CST), Bcl2 (1:1000; Proteintech), Bax (1:1000; Proteintech), Caspase3 (1:1000; Proteintech), The nuclear factor erythroid 2 related factor 2 (NRF2; 1:1000; Proteintech), SOD2 (1:1000; Proteintech) and PKCε (1:1000; Proteintech) overnight at 4°C. Subsequently, the membranes were incubated with appropriate secondary antibodies for 1–2 h. Finally, the blots were visualized by Tanon imaging system (Tanon Science & Technology Co., Ltd., China) and quantified by densitometry using Quantity One software (Bio-Rad, Hercules, CA, United States).

RCC cells treated with sorafenib and/or rHGFK1 were lysed in RIPA buffer with protease inhibitor on ice for 30 min. The supernatant was collected after centrifuging at 13, 000 g for 15 min, and protein content was quantified with a BCA protein concentration kit (Beyotime, Shanghai, China). And then, equal quantities of protein samples were separated with SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% nonfat-dried milk for 1 h at room temperature. Primary antibodies were added and incubated overnight at 4 °C. The next day, secondary antibodies were added and incubated for 1 h at room temperature. Chemical signals were visualized with Tanon imaging system (Tanon Science & Technology Co., Ltd., China).