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. 2011 Dec;179(6):2740–2750. doi: 10.1016/j.ajpath.2011.08.011

Figure 4.

Figure 4

Exogenous expression of EGR-1 could enhance Ras prenylation and membrane association and ERK 1/2 reactivation in Beas-2B cells. A: Beas-2B cells were infected with EGR1 adenovirus infection (Ad-Egr-1) or control adenovirus (Ade). The cells were then lysed and separated into membrane (P100) and cytosolic (S100) portions and subjected to immunoprecipitation and Western blotting against K-Ras and glyceraldehydes-3-phosphate dehydrogenase (GADPH), which was used to show that the separation of membrane and cytosol was successful. The result showed that membrane-associated K-Ras was enhanced by the exogenous expression of EGR-1. B: Beas-2B cells were infected with EGR1 adenovirus infection (Ad-Egr-1) or control adenovirus (Ade). The cell lysates were extracted with Triton-X114, and then the aqueous (upper) and organic (lower) phases were subjected to immunoprecipitation and Western blotting against Pan-Ras, K-Ras, and GAPDH. The GAPDH was used to show that the separation of the aqueous and organic phases was successful. The EGR1 adenovirus-infected cells were also pretreated with FTI, GTI, or vehicle for 48 hours before cell collection. The results showed that prenylated Pan-Ras and K-Ras (lower) were enhanced by exogenous EGR-1 expression. The farnesylation inhibitor FTI has only a slight effect on prenylation, whereas the geranylation inhibitor GTI was able to block the prenylation of both Pan-Ras and K-Ras. This suggested that EGR-1 is able to enhance Ras geranylation. C: Beas-2B cells were co-infected with EGR1 adenovirus (Ad-Egr-1) and a scrambled control or GGPPS siRNA and also co-infected with control adenovirus (Ade). The cell lysate was also subjected to membrane separation through TritonX-114, and then the aqueous (upper) and organic (lower) phases were subjected to immunoprecipitation and Western blotting against K-Ras. EGR-1-enhanced K-Ras prenylation was blocked by GGPPS small-interfering RNA. D: EGR-1 was overexpressed in Beas-2B cells, which was able to activate ERK 1/2. This activation could be inhibited by both GTI and FTI, which suggests that prenylation is crucial to EGR-1-dependent mitogen-activated protein kinase ERK 1/2 activation. E: Beas-2B cells were infected with the DnEGR-1 or control adenovirus and transfected with scrambled or GGPPS small-interfering RNA. After exposure to 10% CSE for 4 hours, the cell lysates were extracted with Triton-X114, and then the aqueous (upper) and organic (lower) phases were subjected to immunoprecipitation and Western blotting against K-Ras. K-Ras membrane binding was increased by 10% CSE treatment for 4 hours while it was decreased by DnEGR-1 and small-interfering GGPPS administration.