Western Blot Assay Protocol

Western blot assay was conducted as previously described^{18 (link)}. Briefly, cells were rinsed with PBS and resuspended in Western and IP Lysis Buffer (P0013, Beyotime) with 1 mM PMSF. The cell supernatants were collected after centrifugation at 12,000 rpm for 5 min. Protein samples were quantified using the BCA Protein Assay Kit (P0012, Beyotime). A total of 40–80 µg proteins were separated using 8–15% gradient SDS-PAGE gels and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). The membranes were then incubated with 5% fat-free milk, primary antibodies (1:1000), and appropriate HRP-conjugated secondary antibodies (1:5000). Protein expression was visualized using an enhanced ECL detection kit (ORT2655, PerkinElmer, Waltham, MA, USA) and GE Amersham Imager 600 (GE, Chicago, IL, USA). The gray values of the bands were analyzed using ImageJ 1.52a.