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. 2011 May 1;509(1-4):16–25. doi: 10.1016/j.abb.2011.02.016

Fig. 2.

Fig. 2

Real-time RT-PCR and Western blot analysis for lipoprotein-induced Egr-1 and Nrf2 expression in endothelial cells in the absence or presence of MAPK inhibitors. (A) Cells were preincubated for 30 min with 25 μM PD98059 or 10 μM SB20358 prior to stimulation with 100 μg/ml lipoprotein for the indicated times. Equal amounts of RNA were subjected to Real-time RT-PCR. Egr-1 mRNA expression level was normalized to GAPDH (∗∗∗p < 0.001). (B) Cells were incubated with 100 μg/ml lipoprotein for the indicated times. (C) Cells were incubated with indicated concentrations of HOCl–HDL for 1.5 h. (D) Cells were incubated with 100 μg/ml lipoprotein for 1.5 h. (E) Cells were preincubated for 30 min with 25 μM PD98059 or 10 μM SB203580 prior to stimulation with 100 μg/ml HOCl–HDL for 1.5 h. (B–E) Cells were lysed and equal amounts of proteins were subjected to Western blot analysis for Egr-1 and Nrf2. After stripping membranes, β-actin was used as a loading control. (F) Cells were preincubated for 30 min with 25 μM PD98059 or 10 μM SB20358 prior to stimulation with 100 μg/ml lipoprotein for the indicated times. Equal amounts of RNA were subjected to real-time RT-PCR. Nrf2 expression level was normalized to GAPDH. (B–E) Lane 1 represents non-stimulated controls, i.e. cells in the absence of lipoproteins. (A–F) One representative experiment (performed in triplicate A, F) out of three is shown.