Detecting the expression of Zeb1, TGF-β, and Smad3 by western blot

After cells were washed 3 times in pre-chilled PBS, cultured cells were lysed in lysis buffer in a culture bottle (100 μl/50 ml) and placed on ice for 30 min, followed by a 10-min centrifugation at 12 000 rpm at 4°C. Then, supernatant was absorbed and placed in a 0.5-ml centrifuge tube separately and saved at −20°C. PBS was used to dilute 2 μg/μl BSA protein standards into 20 μg/μl, 15 μg/μl, 10 μg/μl, 5 μg/μl, 2.5 μg/μl, and 0 μg/μl. Based on the number of samples, we calculated the amount of BCA detection reagent, and blended solution A and solution B (BCA kit, Pierce Co.) at a 50:1 ratio.

A total of 2 μl protein was diluted with 18 μl of double-distilled water and each sample was divided and placed into 2 wells. After 200 ul of detection liquid were added to 96-well plates, diluted protein standards and detecting samples (10 ul/well) were added in detection wells with light and even shaking, followed by 30-min incubation at 37°C and then cooling to room temperature. The absorption value of samples was obtained using a full-automatic microplate reader (Beijing Nuoya Instrument Co.) at 490 nm, and the protein concentration of samples was calculated with a standard curve. All samples were saved at −70°C.

In a cold chamber at 4°C, electrophoresis began at 60 V and then at 120 V for 1–2 h as soon as protein went into separation gel. The wet-transfer method was used to transfer protein onto PVDF membrane for 2 h in the 4°C cold chamber. After PVDF membrane was removed, samples were sealed with 5% nonfat dried milk Tris-buffered saline tween (TBST) for 1–2 h at room temperature. Then, the samples were incubated with primary antibodies for Zeb1, TGF-β, and Smad3 overnight at 4°C, and washed with TBST (10 min ×3). The cells were then incubated with secondary antibodies at room temperature for 1 h and washed 3 times in TBST (10 min ×3). After the samples were exposed and fixed to X-ray film, chemiluminescent assay was performed to detect protein bands, followed by data analysis.