Western Blot Analysis of Protein Samples

Cells were lysed in Laemmli buffer [(100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% glycerol and 200 mM DTT (dithiothreitol)] and then processed for Western blot analysis, as previously described [33 (link),36 (link)]. Briefly, after SDS-PAGE, proteins were transferred on PVDF membrane (Merck-Millipore, Burlington, MS, USA) which was then blocked with 5% milk in PBS for 30 min and then incubated with the appropriate primary antibodies (over-night at 4 °C) and secondary HRP-conjugated antibodies (1 h at room temperature), all prepared in 1% milk in PBS. Chemiluminescence reaction occurred using Clarity or Clarity Max (Biorad, Hercules, CA, USA) and the signal was captured by ChemiDoc MP Imaging Systems (Biorad). Densitometric analysis was performed using Image Lab software (Biorad).