Pvdf film

The total proteins of the cells were obtained using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, CN), and one BCA assay kit (ThermoFisher Scientific, the States) was adopted for protein concentrations assay. The lysates were treated with 10% SDS-PAGE to separate target proteins based on their molecular weight, and the proteins were transferred to the PVDF films (Millipore, the States), followed by blocking in 5% defatted milk. The films were then treated with first antibodies against FGF9 (1 : 1000, Santa, CA, the States) and incubated overnight at 4°C, the GAPDH (1 : 1500, Cell Signaling Technology, Massachusetts, the States), and were then probed with the second antibody.

After treatment, the LS180 cells were harvested, and whole-cell pellets were lysed by sonication in 1x sample buffer (200 mM Tris-HCl at pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 0.2% bromphenol blue, and proteinase inhibitor cocktail) and boiled for 10 min. Equivalent amounts of protein lysates were placed in 8% SDS PAGE gels and separated; then, the protein products were electrophoretically transferred to PVDF films (Millipore, Billerica, MA). After incubation for 60 min with 5% skims milk in phosphate-buffered saline with Tween-20 (PBST), the film was rinsed once with PBST, and the primary antibody was gently rocked overnight at 4°C. Membranes were then rinsed 3 times for 5 min and hatched with anti-rabbit or HRP-conjugated anti-mouse antibody diluted 1 : 5000 at room temperature for 1 h. The blot was rinsed in PBST 3 times and developed by an ECL system (Pierce, ThermoFisher). The blot was performed 3 times, and densitometry was quantitated using NIH ImageJ software.

The proteins of the LL sample were extracted using the method put forward by Li et al. (2017) (link). After the separation of proteins by SDS-PAGE (10 % separation gel and 5 % concentration gel), the target proteins (50 μg) were transferred onto polyvinylidene difluoride (PVDF) films (Millipore, Bedford, MA, America). Then, PVDF films were subjected to blockade in 5 % fat-free milk in Tween 20 (TBS-T) solution (0.1 % Tween-20, 150 mM NaCl, 10 mM Tris, 5 mM KCl, pH 7.4) under ambient temperature and then incubated for 12 h under 4 °C using a variety of different primary antibodies: anti-HIF-1α antibody (1:500), anti-PI3K antibody (1:500), anti-AKT antibody (1:500), anti-p-AKT (Ser473) antibody (1:500), and anti-Actin antibody (1:3,000) (Cell Signaling Technology, Beverly, MA, America). Posterior to cleaning three times (10 min per time) with TBS-T solution, the films were cultivated for 1 h under ambient temperature using anti-rabbit horseradish peroxidase-conjugated second antibody (1:5,000) (Abcam, Cambridge, UK). Then, the films were cleaned thrice in TBS-T solution again and the proteins were visualized using the ECL assay kit on a ChemiDoc MP imaging system (Bio-Rad, Hercules, America). The expressing levels of proteins were subjected to quantification via the Quantity-One program 4.6.2 (Bio-Rad, America) and afterward normalized to actin.