Immunoblot Analysis of Signaling Proteins

The steady-state levels of ERK5, p-ERK5, MEK5, NF-κB, p-NF-κB, IκB-α, p-IκB-α, Vimentin, β-actin, HDAC and GAPDH were determined by immunoblot analysis, as previously described.^{44 (link)} Briefly, blots were incubated overnight at 4 °C with primary rabbit antibody reactive to ERK5 (#3372), p-ERK5 (#3371) or primary mouse antibody reactive to p-IκB-α (#9246; all from Cell Signalling) or primary rabbit antibody reactive to NF-κB (#sc-372), IκB-α (#sc-371) or primary mouse antibody reactive to MEK5 (#sc-135986), Vimentin (#sc-32322), GAPDH (#sc-32233), β-actin (#sc-8432; all from Santa Cruz Biotechnology) or HDAC (#05-614, Merck Millipore Corp, Billerica, MA, USA) or primary rabbit antibody reactive to p-NF-κB (#ab131109; Abcam). Next, immunoblots were incubated with anti-rabbit or -mouse secondary antibody conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at RT. The relative intensifies of protein bands were analysed using the densitometric analysis software Image Lab version 5.1—beta, using a Chemidoc MP Imaging System for acquisition (Bio-Rad Laboratories).