Subcellular distribution of α2-chimaerin in the brain. A, α2-chimaerin rabbit polyclonal antibody showed strong reactivity to the N-terminal and was further purified by affinity chromatography on GST and recombinant α1-chimaerin. Western immunoblot analysis of recombinant α2-chimaerin constructs (shown stained with Coomassie blue) detected GST/α2-chimaerin 1–160 (lane 1), GST/α2-chimaerin 36–160 more weakly (lane 2), and full-length α2-chimaerin (lane 3), but not α1-chimaerin (lane 4), chimaerin C-terminal GAP domain (lane 5), or GST (lane 6). B, α2- and α1-chimaerin protein are distributed in different subcellular fractions in brain: P2, mitochondrial, myelin pellet obtained after centrifugation at 12,000 × g for 10 min; P3, microsomal pellet after centrifugation at 100,000 × g for 1 hr; Sol, cytosolic fraction (100,000 × g supernatant);Spm, synaptic plasma membranes purified from P2 fraction. RacGAP activity was analyzed by GAP overlay assay (left panel) (Manser et al., 1992). White bands show the location of proteins with GAP activity detected as areas of increased hydrolysis of [γ³²P]-GTP-Rac. Corresponding Western immunoblots blots were assayed with C-terminal α1-chimaerin antibodies (Kozma et al., 1996) (middle panel) or α2-chimaerin antibodies (right panel). The positions of α2-chimaerin (45 kDa) and α1-chimaerin (38 kDa) are indicated by arrows.C, Comparison of α1- and α2-chimaerin mRNA expression in cortical layers and corresponding α2-chimaerin immunoreactivity show the specificity of the antibody for the α2-chimaerin isoform in brain sections. C, A,A′, Adult distribution of α1-chimaerin mRNA [counterstaining (A) and in situhybridization (A′) (α1 mRNA)]; B,B′, distribution of α2-chimaerin at P17 [counterstaining (B) and in situhybridization (B′) (α2 mRNA); C, α2-chimaerin immunocytochemistry]. I–VI, Cortical lamina; wm, white matter; str, striatum (α2 Ab).