Uh, 10% horse serum, 100 g / ml G418, 200 g / ml hygromycin, 200 ng / ml doxycyclin, 100 units / ml penicillin and 100 units / ml streptomycin. After selection of 4-6 weeks at 37 in a humidified 7% CO2 G418/hygromycin resistant colonies were isolated and expression of the IkB Signaling transgene by Western blot with anti-syn. We characterized the three cell lines with h Heren levels of expression E46K and w Selected a line. Highly expressed syn E46K with h Heren toxicity t in this study Ver, the cells were in the presence of Dox and media Kept changed every 48 hours. Expression of the transgene and induction of differentiation was obtained by removal and addition of NGF to Dox medium at the same time.
Cell culture and transient transfection of cells N2A N2A E46K synuclein Mice were in Minimum Essential Medium Eagle with 10% heat-inactivated f Fetal K Calf serum and 1% penicillin / streptomycin erg Maintained complements. The cells were Deckgl These grown in 24-well plates at 50% confluency at the MP-470 time of transfection. The cells were then transfected with Lipofectamine ? 2000, according to the manufacturer’s protocol. In short, 0 5 g of DNA, and 0 5 l Lipofectamine ? 2000 in 25 liters of Opti-MEM medium were are diluted. After incubation for 5 min at room temperature, DNA and lipofectamine ? 2000 were mixed and incubated for 20 min at room temperature. 50 l of the mixture of DNA and lipofectamine ? 2000 were added to the cultures. Medium with DMSO or baicalein was replaced 4 h after transfection. The cells were then fixed and the Immunfluoreszenzf Staining within the specified time.
Lyophilized in vitro test aggregation syn E46K protein was dissolved in double-distilled water St, diluted to a concentration of 0 8 mg / ml in PBS and centrifuged at 15,000 g for ? remove aggregated material. A Stamml Solution of 10 mM was prepared in Baicalein 100% dimethyl sulfoxide. Perform experiments for aggregation, 50 M aliquot was mixed with a final concentration in syn baicalein from 0. 1, 1, or 10 M and at 37 without stirring. A control reaction, which only syn E46K and 0 1% DMSO was also prepared. Light scattering measurements An aliquot of 10 L of each aggregation reaction was added to 0. 99 ml of PBS. A Ma of 90 ? light scattering each L solution was taken as 0. Helma QS 5 mm cell using a spectrofluorometer to 3 FluoroMax two wavelengths Lengths of excitation and emission set at 450 nm and a slit width of 1 nm.
The data are determined as the average of three measurements before and after deduction of the buffer signal. Thioflavin T fluorescence Thioflavin T fluorescence assay was performed as described in the samples using a FluoroMax spectrofluorimeter above third After the addition of a final concentration of THT in 50 M and 450 nm excitation, the fluorescence was measured at a wavelength Length of 482 nm emission is measured. The data are determined as the average of three measurements before and after deduction of the buffer signal. Ma took Dichro Sme circular Shaped FUV spectra were collected on a CD spectrometer with a Chirascan Helma QS bowl with a 0. A path length L 5 mm. CD spectra were recorded with a step of 1 nm, a bandwidth of 1 nm and an integration time of 1 s. Before CD analysis, each sample was diluted in PBS syn E46K to a final concentration