Figure 1.

α-Synuclein specifically downregulates PKCδ isoform in N27 dopaminergic cells. A, Whole-cell extracts from stably expressing αsyn N27 cells (Syn), vector control N27 cells (Vec), and rat substantia nigra brain (rSN) were prepared. Expression of αsyn and TH were determined by immunoblotting assay with antibodies against αsyn (Syn-1; BD Biosciences) and TH. β-actin was used as a loading control. B, The specific downregulation of PKCδ protein in αsyn-expressing N27 cells. Representative immunoblots (left panel) and quantitation (right panel) of PKC isoforms (δ, α, βI, and ζ) in whole-cell lysates in αsyn-expressing (Syn) and vector control (Vec) N27 cells. Data shown are mean ± SEM from three separate experiments (***p < 0.001). C, Left, Semiquantitative RT-PCR analysis of mRNA levels of various PKC isoforms. Amplicon base pairs are shown at the right sides of the panel. GAPDH was used as loading control. Right, qRT-PCR analysis for PKCδ mRNA expression in αsyn-expressing and vector control N27 cells. Data shown represent mean ± SEM from four separate experiments performed in triplicate (***p < 0.001). D, Transient overexpression of human wild-type αsyn in N27 cells by lentiviral infection downregulates PKCδ protein expression. N27 cells were infected with lentiviruses expressing LacZ-V5 (control lentiviral vector) or αsyn-V5 for 48 h, and whole-cell lysates were analyzed for V5 and β-actin (top panel), PKCδ (middle panel), and αsyn (bottom panel). A representative immunoblot is shown.