Image pro plus 6.0 software analysis system

Protein lysates were isolated using lysis buffer (Beyotime Biotechnology, Shanghai, China) and quantified by the bicinchoninic acid method (Thermo Fisher Scientific, Waltham, MA, USA). Protein lysates (20 μg) were fractionated onto 10% SDS-PAGE (Sangon) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Whatman, Germany). PVDF membranes were blocked with 5% skim milk-TBS at 4°C overnight, incubated with specific primary antibody solutions at room temperature for 1 hour, and incubated with HRP goat anti-rabbit/ mouse IgG (1:20,000) at room temperature for 40 minutes. Enhanced chemiluminescence system (Millipore, Billerica, MA, USA) and Image-Pro Plus 6.0 software analysis system (Media Cybernetics, Inc., Bethesda, MD, USA) were used for protein blot quantitation. Antibodies against E-cadherin (1:1,000), N-cadherin (1:400), MDM2 (1:1,000), vimentin (1:1,000), β-catenin (1:5,000), Snail (1:500), Slug (1:1,000), Smad2/3 (1:2,000), p-Smad2 (1:1,000), p-Smad3 (1:2,000), and secondary antibodies were purchased from Boster Biotechnology (Wuhan, China). GAPDH antibody (1:10,000) was purchased from Yasunari Biological Engineering (Shanghai, China).

Protein lysates were isolated, quantified, and electrophoretically fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gel (Sangon), and then transferred onto Whatman polyvinylidene difluoride (PVDF) membranes (Millipore, Whatman, Germany) according to standard methods. The membranes were blocked with 5% skim milk and tris-buffered saline (Sigma-Aldrich; Merck KGaA) at 4 °C overnight. Then, the membranes were subjected to primary antibody incubation with antibodies against CDK5 (1:1,000; Cell Signaling Technology, CST, Danvers, MA, USA), ERK1/2 (1:1,000; CST), p-ERK1/2 (1:1,000; CST), PPARγ (1:1,000; CST), p-PPARγ^Ser273 (1:1,000; CST), and GAPDH (1:10,000; Boster Biotechnology, Wuhan, China) at 4 °C overnight. Incubation with secondary antibody (horseradish peroxidase goat anti-rabbit/mouse IgG; 1:20,000; Boster Biotechnology) was conducted at room temperature for 40 minutes. An enhanced chemiluminescence (ECL) system (Millipore) was used for signal detection, and the Image-Pro Plus 6.0 software analysis system (Media Cybernetics, Inc., Bethesda, MD, USA) was used to analyze protein signals. Data were presented as fold change, meaning the expression of the detected protein relative to the control protein. Each detection was replicated for 3 times.