Cortical neurons transduced with AAV.ULK1.DN and AAV.CTRL were lysed and the protein content of each sample was determined as described above. After precipitation with acetone, 50 µg of protein lysates per sample were separated on a 4–12% NuPAGE Novex Bis-Tris Minigel (Thermo Fisher Scientific). Following Coomassie staining, the protein areas were cut out, diced, and subjected to reduction with dithiothreitol, alkylation with iodoacetamide, and finally ON digestion with trypsin. Tryptic peptides were extracted from the gel, the solution dried in a Speedvac, and subjected to nanoLC-MS/MS as described previously [10 (link), 22 (link)]. Details of mass spectrometric analysis and data processing are provided in the SI .
Functional annotation of differentially regulated proteins to GO terms was performed in DAVID version 6.8 [21 (link)]. The functional annotation module was applied for GO biological process and cellular component terms using an EASE score of 0.1 and a minimum number of 2 counts. For analysis of protein–protein interaction networks, STRING version 10.5 [23 (link)] was used with a minimum required interaction score of 0.4. The STRING database’s k-Means clustering tool was employed to group proteins with roles in similar processes into four clusters.
Functional annotation of differentially regulated proteins to GO terms was performed in DAVID version 6.8 [21 (link)]. The functional annotation module was applied for GO biological process and cellular component terms using an EASE score of 0.1 and a minimum number of 2 counts. For analysis of protein–protein interaction networks, STRING version 10.5 [23 (link)] was used with a minimum required interaction score of 0.4. The STRING database’s k-Means clustering tool was employed to group proteins with roles in similar processes into four clusters.
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