Figure 4.

Performance of the modified TRIzol protocol evaluated by immunoblotting. To compare the modified TRIzol protocol versus the TRIzol^® manufacturer’s, mirVana™ PARIS™ kit and standardized laboratory (A+2X Buffers) protocols, 10⁶ HCT116 cells were plated in 14 independent 100 mm plates (one per experimental setting: lanes 1 to 14), and cells were processed for total protein extraction 48 h after plating. Next, 40 μg of total protein extracts were separated on 8% SDS-PAGE and transferred onto nitrocellulose membrane. Immunoblot analysis of steady-state expression levels of housekeeping proteins (β-actin and GAPDH), NF-κB, IκB, p-Akt, Akt, p53, and nuclear PARP.