ArfGAP1 is expressed in the mammalian brain.

<p>(A) Western blot analysis of ArfGAP1 protein levels in mouse brain, with highest levels detected in cerebral cortex (<i>Ctx</i>) and cerebellum (<i>Cb</i>), moderate levels in striatum (<i>Str</i>) and ventral midbrain (<i>VMB</i>), and lowest levels in olfactory bulb (<i>Olf</i>) and spinal cord (<i>SC</i>). LRRK2 is detected at highest levels in striatum, cortex and cerebellum. β-tubulin indicates equivalent protein loading. (B) Subcellular fractionation of mouse whole brain tissue. ArfGAP1 is broadly detected with particular enrichment in membrane-associated fractions including the heavy (P2) and synaptosomal (LP1) membrane fractions, and soluble cytosolic (S1 and S3) and synaptosomal cytosolic (LS1) fractions. LRRK2 (MJFF-2/c41-2) is also broadly detected with enrichment in membrane-associated fractions, including heavy (P2) and light (P3) membrane, synaptosomal (LP1) and synaptic vesicle (LP2) fractions, in addition to soluble cytosolic (S1 and S2) and synaptosomal cytosolic (LS1) fractions. ArfGAP1 and LRRK2 co-associate in the S1, P2, S2, LP1 and LS1 fractions. The distribution of marker proteins demonstrates the enrichment of mitochondria/heavy membranes (TIM23; P2 and LP1), endoplasmic reticulum (PDI; P2, P3, LP1 and LP2), Golgi (Giantin; P2 and P3), synaptosomes/synaptic vesicles (synaptophysin 1; P2, P3, LP1 and LP2) and synaptosomal/synaptic vesicle cytosolic (α-synuclein; LS1 and LS2). (C) Confocal fluorescence microscopy reveals the localization of endogenous ArfGAP1 to the Golgi complex and cytoplasm of MAP2-positive cortical neurons and tyrosine hydroxylase (TH)-positive dopaminergic neurons in rat primary cortical and midbrain cultures, respectively. (D) Validation of short hairpin RNA (shRNA)-mediated silencing of endogenous ArfGAP1 expression in rat primary cortical neuronal cultures. Lentiviral vectors expressing non-silencing shRNA (LV-sh-control) or ArfGAP1-specific shRNAs (LV-sh-ArfGAP1 #1 and LV-sh-ArfGAP1 #2) were used to infect primary neurons at increasing viral doses ranging from 3.3 to 50 ng of P24 antigen per ml of media. Western blot analysis of neuronal extracts reveals the viral dose-dependent knockdown of ArfGAP1 with ArfGAP1-specific shRNAs but not control shRNA. β-tubulin indicates equivalent protein loading. (E) Silencing of endogenous ArfGAP1 expression in GFP-labeled cortical neurons with lentiviral-shRNA vectors revealed by confocal fluorescence microscopy with a rabbit anti-ArfGAP1 antibody. Molecular mass markers are indicated in kilodalton (kDa). Scale bars: 10 µm.