The proteins were separated by electrophoresis and transferred to nitrocellulose mem branes. The membranes were blocked with 5% nonfat dry milk in 0. 01 M PBS and 0. mostly 05% Tween 20 at room temperature for 1 hour. Subsequently, the mem brane was incubated with primary antibodies directed against target proteins overnight at 4 C. The final dilu tions for primary antibodies were 6E10, LC3, and Beclin1. After three quick washes in TPBS, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase diluted at 1 5,000 in TPBS for 1 hour. The immuno complexes were detected by the enhanced chemilumin escence method. The density of immunoblotting was quantified with the software of Quantity One. Assaying AB42 degradation The analysis for AB42 degradation in the NG2 cell line was performed as previously described.

NG2 cell line cultured in Sato medium containing 1% horse serum was exposed to human AB42. AB42 levels in cell culture supernatants and adherent cells were determined by immunoblotting. For some experiments, NG2 cell line was incubated with HiLyte Inhibitors,Modulators,Libraries Fluor 488 AB42 Inhibitors,Modulators,Libraries for 6 hours, followed by the treatment of leupeptin, pepstatin A, wortmannin, and bafilomycin A1 Inhibitors,Modulators,Libraries for 18 hours. The protein levels of AB42, LC3, and beclin1 were determined by immunoblotting. Real time PCR NG2 cell line was incubated with HiLyte Fluor 488 labeled AB42 for the indicated times. Total RNA extraction and reverse transcription for NG2 cell line and cerebral cortex were performed as previously reported with some modifications. Briefly, total RNA was extracted using Trizol reagent.

After treated with RNase free DNase I, first strand cDNA was synthesized Inhibitors,Modulators,Libraries with M MLV reverse transcript ase and Oligo dTs. Real time quantitative PCR was conducted with ABI Prism 7500 Sequence Detection System according to the instruction of the manufacturer. Dissociation curve analyses were performed using the instruments default setting Inhibitors,Modulators,Libraries immediately after each PCR run to ensure specificity. The expression level of target genes was normalized to the actin gene. The primers which were used for real time PCR are provided in the following Table 1. Small RNA interference Small interfering RNAs were synthesized by Shanghai GenePharma Co The pairs of siRNA oligo nucleotides were prepared as a 20 uM stock.

For transient necessary transfection, NG2 cell line was cultured in six well plates to 80% confluence and transfected with Lipofectamine RNAiMAX according to the manufacturers instructions. After transfection, the cells were left for another 24 hours before they were used for experiments. Quantification of the mCherry LC3 puncta Human microtubule associated protein The nucleotide sequence was inserted into mCherry C1 vector at the Hind III and BamH I restriction sites. The plasmid was verified by sequencing.