Fig. 6.

Effects of EFV on IkBa phosphorylation and NFkB nuclear translocation. (A). The protein levels of total IκBα and phospho-IκBα in the cytoplasmic extract, and nucleus-translocated NFκB in the nucleus extract were determined by western blot analysis. Loading efficiencies for cytoplasmic extract and nuclear extract were determined by reprobing the blot with antibodies against β-actin and laminin, respectively. Specific antibody against NFκB subunit p65 was used to detect this protein in the whole cell extract. Relative density of the protein bands of phospho-IκBα was normalized to β-actin as the fold of the control. (B) The protein levels of NFkB in the nucleus extract were determined by western blot analysis. Loading efficiencies were determined by reprobing the blot with the antibody against β-actin. Specific antibody against NFkB subunit p65 was used to detect this protein in the nuclear extract. Antixodant MnTBAP blocked the effect of EFV. TNF-α was used as a positive control. Relative density of the protein bands of NFκB (p65) was normalized with corresponding β-actin bands. (C) HCAECs were infected with an adenovirus containing a dominant negative form of IκBα (IκBα-DN-Adv) for 48 hrs. Cytoplasmic extracts were purified using NE-PER Nuclear and Cytoplasmic Extraction Reagents. Expression of IkBα was determined by western blot analysis. Loading efficiencies were determined by reprobing the blot with the antibody against β-actin. (D) HCAECs were infected with IκBα-DN-Adv (inhibition of IκBα) or GFP-Ad-GFP as a negative control for 48 hrs and the nuclear extract was prepared. Nuclear NFkB (p65) levels were determined by western blot analysis. For EFV effects, HCAECs expressing IkBα-DN-Adv were treated with EFV (10 µg/ml) for 45 min. Loading efficiencies were determined by reprobing the blot with the antibody against β-actin. Relative density of the protein bands of NFκB (p65) was normalized with corresponding β-actin bands as the fold of control.