Western blot

We used radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, PRC) to lyse snap-frozen tumor and corresponding paracarcinomatous tissues, and the bicinchoninic acid protein assay was employed to estimate protein concentration. The same amount of protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to the polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with rabbit primary antibodies anti-PLCE1 (1:300, Abcam PLC, Cambridge, UK) and human antibody of β-actin (1:5,000, Zhongshan Golden Bridge Biotechnology Co Ltd, Beijing, PRC). Following a wash with Tris-buffered saline/0.1% Tween for three times, each time for 5 minutes, we incubated the membranes with secondary antibodies at room temperature for 2 hours. The blots were captured and visualized by Alpha-EaseFC imaging system (Alpha Innotech, San Leandro, CA, USA). Using the Alpha-EaseFC software, the integrated density value (IDV) of each band was detected by drawing a rectangle outlining the band. A total IDV by summation of each band IDV was employed when a protein had double bands. Results were normalized to the internal control, β-actin.