Figure 1.

Expression profile of Ras-GRF1 and Ras-GRF2 in brain extracts and primary striatal cultures of wild-type (WT) and Ras-GRF1 knockout (KO) mice. (A) Brain extracts from WT (+/+) and Ras-GRF1 KO (−/−) animals killed at four different time points were prepared for Western blot (WB) analysis and probed with an antibody against p140^Ras-GRF1. (B) Same extracts as in (A), probed with p135^Ras-GRF2 antibodies. Expression of both Ras-GRF1 and Ras-GRF2 gradually increases over time: postnatal day P0, P7, P14, and P30. Equal amounts of proteins (30 μg) for each time point were loaded. Ras-GRF1 KO and WT mice showed equivalent levels of Ras-GRF2 immunoreactivity in striatum (Str), cortex (Cx), and hippocampus (Hp) at each time point. (C) Representative immunoblots from Str, Cx, and Hp of adult (P60) WT and Ras-GRF1 KO mice. Both Ras-GRF1 and Ras-GRF2 seemed to be highly expressed in Cx and Hp and to a lesser extent in the Str of WT mice. No obvious alterations in the Ras-GRF2 levels could be seen in extracts from Ras-GRF1 KO mice. The p44^ERK1 and p42^ERK2 kinases were also equally expressed in mice of the different genotypes. (D) Protein extracts were prepared from primary embryonic (E16) striatal cells or postnatal (P1) striatal cultures and analyzed by WB. The p140^Ras-GRF1 was only expressed in WT postnatal cells, whereas equal levels of p135^Ras-GRF2 were found in postnatal WT and KO cells. Expression of neurofibromin (NF1), SOS-1, p44^ERK1, and p42^ERK2 are neither developmentally regulated nor affected by the Ras-GRF1 mutation. Tubulin was used as loading control. *Nonspecific band. ERK, extracellular signal-regulated kinase.