Western Blot Analysis of Protein Expression

OS cells, with the applied genetic modifications, were harvested using the described lysis buffer [32 (link)]. Twenty μg lysate proteins per sample were separated by 10- 15% SDS gels, and transferred onto PVDF blots (Millipore, Shanghai, China). The blots were blocked, probed with applied primary and second antibodies [32 (link)]. The enhanced chemiluminescence (ECL) detection system was applied to visualize the targeted protein bands (based on the molecular weights), using x-ray films. For all the Western blotting assays, each lane was loaded with exact same amount of quantified protein lysates, then the same set of lysate samples were run in parallel (“sister”) gels. The ImageJ software was utilized for data quantification [33 (link), 34 (link)].