Peroxide lumiglo reagent

Cells were lysed with sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, China) and denaturalized in a water bath at 100 °C. The prepared protein was resolved via SDS-PAGE, transferred to a polyvinylidene difluoride membrane from Millipore, and incubated with PDZRN4 primary antibody and HRP-Goat Anti-Rabbit IgG (H + L) (MultiSciences, Hangzhou, China). Protein levels were detected with a peroxide LumiGLO reagent (Cell Signaling Technology, 7003). Quantification of the relative protein levels was conducted according to the ratio of gray-value to the corresponding internal references. A representative graph was constructed and protein was quantified based on five pairs of clinical samples or three independent experiments and shown as mean ± SD.

The cultured cells were lysed with the sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, Shanghai, China) on ice for 10 min and centrifuged at 4 °C for 15 min at the maximal speed. The supernatants were collected into new Eppendorf tubes for assessment of the concentration using the enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime) and denaturalized in a water bath at 100 °C for 5 min. Equal amounts of the protein samples were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) followed by Western blotting. The membranes were incubated with anti-MDM2 (Abcam, Cambridge, MA, USA), anti-MT1M (Abbexa, Cambridge, UK), anti-p53 (Abcam), anti-phosphorylated p53 (Abcam), anti-MMP14 (Abcam), anti-MMP9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP2 (Abcam) or anti-β-actin (MultiScience, Hangzhou, Zhejiang, China) antibody and then with the corresponding secondary antibodies according to the manufacturer’s Western blot protocols. The relative protein levels were detected with a peroxide LumiGLO reagent (Cell Signaling Technology, Danvers, MA, USA) and quantified according to the ratio of the gray value to the corresponding β-actin levels. The experiments were repeated three times independently.