Torin 1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Potent and selective mTOR inhibitor that Displays 200-fold selectivity for mTOR over DNA-PK, ATM and hVps34	2nM and 10nM concentration of Torin1 to be used to induce autophagy.

Upstream tips
Potent and selective mTOR inhibitor that Displays 200-fold selectivity for mTOR over DNA-PK, ATM and hVps34
Protocol tips
2nM and 10nM concentration of Torin1 to be used to induce autophagy.

Publication protocol

Standard western blotting protocol was followed (Mahmood and Yang 2012). Cells were lysed in ice-cold RIPA buffer by incubating in ice for 30 min and centrifuged at 16,000g for 15 min. Supernatant was collected and protein estimated by the Bradford method (Bradford 1976). 30 μg protein was loaded onto gels (10% or 15%) and transferred onto PVDF membrane. The membranes were blocked with 5% skimmed milk in Tris-Buffered Saline containing 0.5% Tween-20 (TBST) and incubated overnight with primary antibody (anti-α-syn [1:30,000], anti-LC3 [1:1000], anti-p62 [1:50,000], anti-beta actin [1:2500], anti-Histone [1:500]). Membranes were then probed with HRP-tagged secondary antibody (1:10,000 or 1:30,000), bands detected using Chemiluminiscence (Millipore) and captured on X-ray films. The X-rays films were imaged using Uvipro Platina (UVItec Gel documentation system) and densitometry analysis was performed using ImageJ analysis software. The bands were quantified and normalized with their respective β-actin.

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Manufacturer protocol

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