β tubulin

Proteins were extracted from cells by using RIPA Lysis Buffer (Beyotime, China) supplemented with a cocktail of protease inhibitors (Beyotime, China) after rinsing cells three times with cold PBS. The lysates were then subjected to centrifugation at 12,000×g at 4 °C for 5 min. Next, the isolated protein samples were mixed with sample buffer and boiled for 5 min at 100 °C. The total protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). Equivalent amounts of protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Sigma, USA). After blocking with 5% skimmed milk, the membranes were incubated with the following antibodies: MMP17 (1:1000, Zenbio), SH3GL2 (1:1000, Zenbio), and β-Tubulin (1:1000, Solarbio). The membranes were then incubated with appropriately HRP-conjugated secondary antibodies (1:5000) and extensively rinsied with TBST. Immunoreactive bands were detected using a chemiluminescence kit (ECL Substrate kit; Abclonal, China), and the protein bands were captured using the Amersham Imager 6000 (GE Healthcare).