Rabbit anti lamp2

The mouse anti-APP, mouse anti-KIF3B and rabbit anti-ULK1 were purchased from Cell Signaling. Rabbit anti-Atg16L1 and rabbit anti-Atg5 were purchased from ABclonal (China). Rabbit anti-β-actin, rabbit anti-LC3B-Specific, rabbit anti-P50, rabbit anti-DHC1, rabbit anti-DlC3 and rabbit anti-P62/SQSTM1 were purchased from Proteintech (US). Mouse anti-DIC was bought from Millipore. Rabbit anti-RILP and rabbit anti-Lamp2 were purchased from Abcam. Mouse anti-Huntingtin and mouse anti-BICD2 were purchased from Santa Cruz. All the secondary antibodies were purchased from Bioworld Technology.

pAAV-SYN-MCS-EGFP-3FLAG-miR30shRNA(Dync1i2), pAAV-SYN-Dync1i2-2A-EGFP-3FLAG, pAOV-SYN-MCS-EGFP-3FLAG and pAAV-SYN-MCS-EGFP-3FLAG were from Obio Technology (Shanghai, China). The plasmids si-m-Dync1i2_001(CGAAGAGAATGATAGCAAA), si-m-Dync1i2_002(GGTGCTAAGCTGTCATTAA), si-m-Dync1i2_003(GGACAACTAAGAATAACAA), si-m-NC were constructed by Ribo Biotechnology Company(Guangzhou, China). Rabbit anti-β-actin, rabbit anti-LC3B-Specific, rabbit anti- P50, rabbit anti-KIF5B and rabbit anti-P62/SQSTM1 were purchased from Proteintech (US). Mouse anti-P150 was tought from BD. Mouse anti-DIC was bought from Millipore. Mouse anti-Rab7 and rabbit anti-Lamp2 were purchased from Abcam. Mouse anti-4G8(β-Amyloid, 17-24) was bought from Biolegend. All the secondary antibodies were purchased from Bioworld Technology.

After treatments, the cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (Beyotime, Beijing, China) with protease inhibitors, sonicated for 15 seconds and centrifuged at 12,000 g for 15 minutes at 4 °C. The protein concentrations were determined by a BCA protein assay kit (Beyotime, Beijing, China), and then the protein samples were mixed with loading buffer and heated at 100 °C for 10 minutes. Equal amounts of protein samples were loaded per lane, separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore), which were then blocked with 5% non-fat milk for 3 hours. The PVDF membranes were then probed with different antibodies: rabbit anti-LAMP2 (1:1500, Abcam), rabbit anti-Beclin1 (1:1000, CST), rabbit anti-LC3 (1:1000, CST), rabbit anti-cleaved-caspase3 (1:1000, CST) and mouse anti-β-actin (1:5000, Sungene Biotech). After the primary antibody binding, the membranes were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit (1:8000, MultiSciences, GAR007, China) or goat anti-mouse IgG (1:8000, MultiSciences, GAM007, China) at room temperature for 1 hour. The reactive proteins were detected by an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA), the band intensities were quantified with Quantity One software, and the results were normalized to β-actin.