Effects of sCD40L on NF-κB activation in HCAECs. (A) IκBα phosphorylation. HCAECs were treated with sCD40L for different time points, and phosphorylation levels of IκBα were determined by a Bio-Plex Luminex Immunoassay with a commercial kit. U test. (B) NF-κB p65 protein nuclear translocations. HCAECs were infected with recombinant adenovirus Ad-IκB DN (inhibition of IκBα) or Ad-GFP (a negative control). The cells were then treated with sCD40L for different time frames, and nuclear extract was prepared. Nuclear NF-κB protein levels were determined by Western blot analysis. Nuclear protein laminin was included as a loading control. (C) Effect of dominant negative mutant form of IκBα on eNOS mRNA levels. HCAECs were infected with recombinant adenovirus Ad-IκB DN or Ad-GFP for 72 hours, and then cells were treated with sCD40L for 24 hours. The NOS mRNA levels of eNOS were determined by real-time PCR analysis. U test. (D) Effect of dominant negative mutant form of IκBα on eNOS protein levels. HCAECs were infected with recombinant adenovirus Ad-IκB DN or Ad-GFP for 72 hours, and then cells were treated with sCD40L for 24 hours. The eNOS protein levels of eNOS were determined by Western blot analysis. *P < .05; **P < .01 compared with the control. n = 3. Data are means and SE of multiple experiments (n).