After release from quiescence in the presence of DMSO or GUTK, cells were harvested and treated with ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail. Protein quantification, electrophoresis and western blotting were performed as previously described.42 (link) Antibodies (human specific) used were c-MYC (Abcam, Epitomics, Cambridge, UK, #1472-1), S62-phospho-c-MYC (ab51156), T58-phospho-c-MYC (ab28842) and GAPDH (ab128915) were obtained from Abcam Company (Cambridge, UK). Additional antibodies were phospho-Rb (Ser807/811) (#9308), ERK1/2 (137F5) (#4695), phospho-ERK1/2 (Thr202/Tyr204) (#4370), GSK3β (27C10) (#9315) and phospho-GSK3β (Ser 9) (#9323) purchased from Cell Signaling Technology (Danvers, MA, USA), FBXW7 (A301-721A) were obtained from Bethyl Laboratories (Montgomery, TX, USA) and α-tubulin (sc-5286) from Santa Cruz Biotechnology (Dallas, CA, USA).
Immunoblot images were exported in the format of tagged image file and quantified using ImageJ 1.46 software (National Institute of Health). After normalization to loading control, the immunoblot bands of c-MYC and FBXW7 were plotted to determine the half-life. The 50% decrease in protein intensity based on the Y-axis was used to determine the corresponding value on the X-axis, which by definition is the time required for the proteins of interest to decrease to the half of the baseline levels.
Immunoblot images were exported in the format of tagged image file and quantified using ImageJ 1.46 software (National Institute of Health). After normalization to loading control, the immunoblot bands of c-MYC and FBXW7 were plotted to determine the half-life. The 50% decrease in protein intensity based on the Y-axis was used to determine the corresponding value on the X-axis, which by definition is the time required for the proteins of interest to decrease to the half of the baseline levels.
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