Ecl808 25

The total protein was extracted using a RIP Assay Kit (R0010, Beijing Solarbio Life Sciences, Beijing, China). The protein concentration was measured using a bicinchoninic acid Kit (G3522-1, GBCBIO Technologies, Guangzhou, Guangdong, China). Based on the different concentrations, the proteins were quantified, separated by polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane using a wet transfer method, and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Then diluted primary antibodies rabbit polyclonal antibodies to vimentin (1:1,000, Abcam, Cambridge, UK), N-cadherin (1:1,000, Abcam, Cambridge, UK), and E-cadherin (1:1,000, ab76055, Abcam, Cambridge, UK) were added for incubation overnight at 4°C. After that, the membrane was incubated with horseradish peroxidase-labeled goat-anti-rabbit IgG antibody (1:5,000, Beijing Zhongshan Biotechnology, Beijing, China), and reacted with enhanced chemiluminescence solution (ECL808-25, Biomiga, San Diego, USA) at the room temperature for 1 min. The anti-GAPDH (1:1,000, ab8245, Abcam, Cambridge, UK) antibody served as the internal reference. The relative levels of proteins were calculated by the ratio of the gray value between the target band and the internal reference band.

Western blot assay was performed to determinate the protein expression levels of ERK, JNK, p38, BAX and Bcl-2 as previously described [18 (link)]. The tissues and cells were lysed with lysis buffer and centrifuged at 12000 r/min for 30 min at 4 °C to collect total protein. The protein (50 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane, which was then blocked using 5% skimmed milk for 1 h at room temperature. Afterwards, the membrane was incubated with the following primary antibodies at 4° overnight: ERK (1: 1000, ab196883), JNK (1: 1000, ab4821), p38 (1: 1000, ab170099), BAX (1: 2000, ab32503), Bcl-2 (1: 1000, ab32124), p-ERK (1: 200, ab214362), p-JNK (1: 1000, ab124956) and p-p38 (1: 1000, ab4822). All antibodies were procured from Abcam Inc. (Cambridge, MA, USA). Next, the membrane was incubated with horseradish peroxidase (HRP)-labelled secondary antibody for 1 h. Subsequently, the membrane was developed with enhanced chemiluminescence (ECL) solution (ECL808-25, Biomiga, San Diego, CA, USA) for 1 min at room temperature. Resulting protein bands were observed using an X ray machine (36209ES01, Shanghai Qcbio Science & Technologies Co., Ltd., Shanghai, China).