H4A3

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For WB use concentration of 0.2-0.5 µg/ml for primary Ab

Protocol tips
For WB use concentration of 0.2-0.5 µg/ml for primary Ab

Publication protocol

After treatment for 48 h, nucleofected cells in 60 mm dishes were incubated for seven minutes at 37°C with Accutase (Innovative Cell Tech., AT104) to detach cells from their substrate. Whole cell lysates were collected and protein concentrations were determined using BCA protein assay (Fisher Scientific, PI-23227) as previously described [8]. Equal amounts of each protein sample were then electrophoresed using 12% Tris/Glycine SDS-polyacrylamide gels, or for APP CTFs using 16.5% Tris-Tricine pre-cast gels (Bio-Rad Laboratories, 456–3064) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, 162–0177). Following a 30 minute incubation at room temperature with 5% blocking milk (Bio-Rad, 170–6404) in 1X tris-buffered saline with tween (TBST), membranes were incubated overnight with primary antibodies for immunodetection of the following proteins: LAMP1 (University of Iowa Hybridoma Bank, H4A3); LC3 (rabbit anti-LC3, Abcam, ab51520); α-syn (Santa Cruz Biotechnology, Inc., SC7011); and APP CTF (Covance Inc, SIG-39152). Membranes were washed with 1X TBST containing 0.1% Tween 20 and then incubated with secondary IgG-HRP conjugated antibody for 1 h at room temperature. After washing blots, SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, PI-34096) was used to detect α-syn and Enhanced chemilumescence (ECL; Thermo Scientific, PI-32106) was used to detect LAMP-1, LC3 and APP. Blots were then stripped with Restore Western Blot stripping buffer (Thermo Scientific, 21059) and probed for actin (Sigma, A1978) to normalize for gel loading. Films with detected bands of interest were scanned using Adobe Photoshop and band intensities were calculated using UN-SCAN-IT gel digitizing software (Silk Scientific, Inc.).

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Manufacturer protocol

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