Immunoblot Analysis of Protein Dynamics

Immunoblot (IB) experiments were performed as described previously (Jones et al., 2004 (link), 2007 (link); Balut et al., 2010a (link),b (link); Gao et al., 2010 (link); Bertuccio et al., 2014 (link); Farquhar et al., 2017 (link)). Briefly, cells were lysed and protein concentrations were determined by the BCA protein assay (Walker, 1994 (link)). Equal amounts of protein (30 μg) were loaded into wells of a gel (6 or 8%) and protein standard (8 μl) used (BenchMark™ pre-stained protein ladder; Invitrogen, Cat No. 10748-010) and resolved with SDS-PAGE for 150 mV for 90 min (Hoefer Mighty Small II system, Cat. No. 80-6149-35, Amersham Biosciences Corp. Piscataway, NJ, USA). Proteins were transferred (50 V, 2 h) with a semi-dry transfer unit (Hoefer, EPS 2A200) to polyvinylidene difluoride (PVDF) membranes for further IB analysis with α-streptavidin antibody. Proteins bands were visualized by enhanced chemiluminescence detection (Lumilight, Roche, Basel Switzerland). Blots were probed for β-actin as a protein loading control. The bands obtained from immunoblot analysis were quantified by densitometry, using the GS-700 densitometer (Bio-Rad) and the Quantity One programme (BioRad laboratories). The obtained band intensities for the various time points were normalized to β-actin and then compared relative to the intensity at time 0 (t = 0) and reported.