LAMP1 (C54H11) Rabbit mAb

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	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Publication protocol

Western Blot Analysis of p38α MAPK, BACE1, and Autophagy
The frozen brain or cell pellets were homogenized in RIPA buffer. For the detection of phosphorylated p38α MAPK, the extra phosphatase inhibitors (5 mm NaF, 1 mm Na3VO4, 1 mm EGTA, 50 nm okadaic acid, 5 mm sodium pyrophosphate) and 1 mm DTT were added to the RIPA lysis buffer. The proteins were separated with 10 or 12% SDS-PAGE. Rabbit polyclonal antibodies against phosphorylated (Thr-180/Tyr-182) and total p38α MAPK (catalog numbers 9211 and 9212, respectively; Cell Signaling Technology), rabbit monoclonal antibodies against BACE1, LC3B, beclin1, ATG5, and lysosomal membrane-associated protein 1 (LAMP-1) (clone D10E5, D11, D40C5, D5F5U, and C54H11, respectively; Cell Signaling Technology), and rabbit polyclonal antibody against Gm2 activator protein (Gm2A) (Thermo Scientific, Darmstadt, Germany) were used as the primary antibodies in the Western blot. To confirm the results from Western blot with D10E5 antibody, mouse monoclonal antibody against human/mouse BACE-1 ectodomain (clone number 137612; R&D Systems Inc., Minneapolis, MN) was used. For all Western blot analyses described above, α-tubulin or β-actin were detected as a loading control with the DM1A antibody (Abcam, Cambridge, United Kingdom) and the 13E5 antibody (Cell Signaling Technology).

Preparation and Characterization of Lysosome
The procedure was processed as described (35, 36) with minor modifications. Cultured SH-SY5Y cells were harvested by trypsinization and washed with PBS. All subsequent manipulations were carried out at 4 °C using pre-cooled reagents. Washed cells were homogenized in the HB buffer (0.25 m sucrose, 10 mm Hepes, 1 mm EDTA, (pH 7.4)) supplemented with protease inhibitor mixture (Roche Applied Science) at 1 × 108/ml. Homogenates were spun at 800 × g for 10 min to pellet nuclei and unbroken cells, which were then re-homogenized in a half-volume of HB. The two supernatants were pooled, incubated for 10 min at 37 °C in the presence of 2 mm CaCl2, and then centrifuged at 3,000 × g for 10 min to remove large heavy mitochondria. The resultant supernatant was centrifuged for 10 min at 18,000 × g, obtaining a pellet that was re-suspended in 0.5 ml of HB buffer and layered on 4 ml of iso-osmotic Percoll (GE Healthcare, München, Germany) at a concentration of 30% (pH 7.4). Under Percoll, 0.5 ml of 2.5 m sucrose was laid. Centrifugation was performed at 4 °C for 40 min at 44,000 × g. The subsequent gradients were carefully collected with 0.9 ml/fraction into tubes from the top. Protein concentrations were determined and Western blot analysis was performed using antibodies against the lysosomal marker, LAMP-1 (clone C54H11), and non-lysosomal markers, calnexin (rabbit polyclonal to Calnexin; catalog number ab22595, Abcam) and β-actin (clone 13E5).

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