Figure 1.
Generation and characterization of forebrain-specific constitutively active and dominant-negative Rap2 transgenic mice. A, Schematic of Rap2V12 and Rap2N17 transgene constructs. αCaMKII promoter was used to drive expression. HA, Hemagglutinin epitope tag. SV40 intronic sequence and a poly(A) tail were added to the 3′ end of the transgene. B, Brain regions from 2-month-old Rap2V12, Rap2N17, and wild-type littermate mice were immunoblotted with HA or Rap2 antibodies. Last four lanes, Extracts from HEK293T cells expressing HA-Rap2V12 or HA-Rap2N17 were titrated and run alongside the brain extracts. The arrows indicate transgenic Rap2 protein; the arrowheads indicate endogenous Rap2. CTX, Cortex; HIP, hippocampus; CBL, cerebellum; BS, brainstem. C, Immunohistochemistry of brain sections from 2-month-old Rap2V12, Rap2N17, and wild-type littermate mice using antibody against HA. HIP, Hippocampus; STR, striatum; AMYG, amygdala; CBL, cerebellum. Scale bar, 500 μm. D, Rap2 activity assay. Forebrains from 2-month-old Rap2V12, Rap2N17, or wild-type littermate mice were solubilized and incubated with GST-RalGDS RBD. As positive control, WT lysate was preincubated with nonhydrolyzable GTPγS. As negative control, WT lysate was preincubated with GDP. Proteins precipitated by GST-RalGDS RBD were immunoblotted for Rap2, HA, and Rap1. The arrows indicate transgenic Rap2 protein; the arrowheads indicate endogenous Rap2. E, Biochemical fractionation of endogenous and transgenic Rap2V12. Forebrains from 2-month-old Rap2V12 mice were fractionated by differential centrifugation and immunoblotted with Rap2 antibody. Twenty micrograms of protein were loaded for each fraction. F, Developmental expression of Rap2V12 transgenic protein. Forebrain extracts from 1-d-old and from 0.5-, 1-, 2-, 3-, 4-, and 6-month-old mice (Rap2V12 vs wild-type controls) were immunoblotted using HA, Rap2, and tubulin antibodies.