Labworks gel imaging and analysis system

Total protein of MLFs and lung tissues were collected by using RIPA lysate buffer, and the protein concentration of each sample was determined by the BCA method. Then, SDS-PAGE was performed and the proteins were transferred from the gel onto PVDF membranes. Following blocking with 2% BSA, the membranes were incubated with primary and secondary antibodies. The LabWorks™ gel imaging and analysis system (UVP, USA) was used for photography. The band brightness of the samples to be tested was compared with the relative β-actin (internal reference) luminance value, and the ratio obtained was plotted after standardization. Anti-CXXC5, anti-α-SMA, anti-Colla I, anti-CD40L (PE), anti-CD40 (FITC) antibodies, etc. were all purchased from Abcam Company of the United States.

GC cells were lysed with RIPA buffer (Beyotime, Jiangsu, China) in an ice bath for 30 min. The lysate was centrifuged at 12,000 rpm for 20 min, and the supernatant was collected. The protein samples in the supernatant were denatured, separated by 10% sodium dodecyl sulfate -polyacrylamide gel-electrophoresis, and transferred onto polyvinylidenedifluoride membranes (Millipore, MA, USA). The membranes were then blocked with 5% skim milk for 1 hand incubated with primary antibodies against YES1, Ki-67, cyclinD1, survivin, E-cadherin, vimentin, Snail, MMP2, MMP3 and SOX4 (1:200, Saierbo, Tianjin, China) at 4 °C overnight. Afterward, the membranes were washed with Tris-buffered saline and incubated with secondary horseradish peroxidase (HRP-) labeled goat anti-rabbit IgG antibodies (1:10,000, Saierbo, Tianjin, China) for 2 h. β-tubulin (1:500.Saierbo, Tianjin, China) was used as control. The protein bands on the membranes were then detected using Western Lightning Chemiluminescence Reagent (PerkinElmer, USA) and photographed through LabWorksGel imaging and analysis system (UVP, USA).