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Characterization of PC12 cell expressing WT or mutant LRRK2.

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posted on 22.10.2013, 03:14 by Rossana Migheli, Maria Grazia Del Giudice, Ylenia Spissu, Giovanna Sanna, Yulan Xiong, Ted M. Dawson, Valina L. Dawson, Manuela Galioto, Gaia Rocchitta, Alice Biosa, Pier Andrea Serra, Maria Teresa Carri, Claudia Crosio, Ciro Iaccarino

(A) PC12-derived cells expressing myc-tagged human WT or mutant-LRRK2 were left untreated (−) or treated (+) for 48 h with 0.2 µg/mL doxycycline to induce expression of transgenic LRRK2. Cell lysates were subjected to reducing SDS/PAGE. The anti-myc antibody was used to visualize LRRK2 and anti-TH for tyrosine hydroxylase. β-actin serves as controls for equal loading of samples. (B) Dose-dependent expression of doxycyline-inducible LRRK2G2019S. Cells were treated for 48 h with the indicated concentration of doxycycline and equal amounts of protein were tested in Western blot analysis with anti-myc or anti-β-actin antibodies. (C) Effect of doxycycline on DA and DOPAC+HVA concentration in PC12 cell lysates and extracellular medium after 48 h expression. At the beginning of each experiment, 105 PC12 cells/cm2 were plated and after the desired incubation period, the medium was aspirated from each well and stored, and the cells were collected in metaphosphoric acid. Samples were subsequently analyzed for levels of DA and its metabolites DOPAC and HVA in cell lysates and incubation medium. Results are the means ± SEM of three experiments performed in triplicate.

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