Figure 3.

Thrombin-induced inflammatory responses are mitigated in the presence of ER stress inhibitor 4-PBA. HPAEC were pretreated with 4-PBA (10 mM) for 1 h followed by treatment with 5 U/ml thrombin for 6 h. Total cell lysates were probed for BiP/GRP78 (A). HPAEC were transfected with NF-κB-LUC and pTKRLUC constructs by DEAE-dextran as described in Methods. Cells were then treated with 4-PBA (1 h) followed by thrombin treatment (5U/ml) for 6 h and cell extracts were prepared and assayed for Firefly and Renilla luciferase activities (B). Total cell lysates treated with 4-PBA followed by thrombin treatment were also probed for VCAM-1(C) and ICAM-1(D) by western blotting and cell supernatants were analyzed for IL-6 (E) and IL-8 (F) levels by ELISA. The blot in 3 C is cropped from two different parts of the same gel, as shown by white space.