Figure 3. (A-B) Knockdown of Beclin1 inhibits RelA/p65 nuclear DNA binding without affecting IκBα degradation.

HPAEC were transfected with si-Con or si-Beclin1 48 h prior to treatment with thrombin for 1 h. (A) An ELISA-based assay was used to assess RelA/p65 DNA binding activity as described in the Materials and Methods. Bars indicate mean ± SEM (n = 3) for each condition, and were analyzed by ANOVA. (B) Total cell lysates were prepared and Western blots were performed to determine IκBα and RelA/p65 levels. Bars indicate mean ± SEM (n = 3) for each condition, and were analyzed by ANOVA. (C-F) Knockdown of Beclin1 prevents RelA/p65 nuclear translocation via inhibition of cofilin1 phosphorylation and actin stress fiber formation. HPAEC were transfected with si-Con or si-Beclin1 48 h prior to treatment with thrombin for 1 h. (C) Nuclear extracts were analyzed by immunoblotting for RelA/p65 and Lamin B levels. Bars indicate mean ± SEM (n = 3) for each condition, and were analyzed by ANOVA. (D) Total cell lysates were analyzed by Western blot to determine the phosphorylation status of cofilin1 at Ser³. Total levels of cofilin1 were used to monitor protein loading. Bars indicate mean ± SEM (n = 3) for each condition, and were analyzed by ANOVA. (E) Cells were fixed and stained with Phalloidin-488 (green) to mark actin filaments and DAPI (blue) to mark nuclei. Each image is representative of three experiments. (F) Mean fluorescence intensity of actin stress fibers (phalloidin staining)/cell was determined by analyzing 35-39 cells. Values are reported in arbitrary units (AU), and were analyzed by ANOVA.