Autophagy Inhibitor, 3-MA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
It is an autophagosome inhibitor.	Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line

Upstream tips
It is an autophagosome inhibitor.
Protocol tips
Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line

Publication protocol

Drug- or vehicle-treated cells, or mouse tissue samples were lysed in a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3VO4, 0.5 μg/mL of leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime). Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The samples were then scraped and transferred into microfuge tubes, centrifuged at 12,000 rpm for 15 min, and heated in an SDS-PAGE protein loading buffer (Beyotime) at 95 °C for 10 min. Equal amounts of protein were separated on 10 or 15% SDS-PAGE gels (Beyotime). After the electrophoresis, the separated proteins were transferred to a PVDF membrane (Beyotime); the membranes were then blocked in 5% of nonfat milk for 60 min. Next, the membranes were incubated overnight at 4 °C with the following primary antibodies raised against: phospho-GSK3β (Ser9) (#5558), phospho-BTK (#5082P), BTK(#8547), phospho-Akt(#9271), Akt (#9272), LC-3A/B (#12741), Atg (#8558), cyclin D1 (#2922), p-Rb (#3590), p-mTOR(#5536), mTOR(#2972), p-ULK1(#12753), ULK1(#8054), p-p70S6K (#9208), p70S6K(#14130), cleaved caspase 3 (#9661), cleaved caspase 9 (#9502), and Bcl-xL (#2764), from Cell Signaling technology (Danfoss, USA); GAPDH (AG019), from Beyotime; or E2F1 (ab179445), from Abcam (Cambridge, UK). Following a 1-h incubation with horseradish peroxidase (HRP)-labeled secondary antibodies, the blots were developed using a western blot chemiluminescence reagent system (Perkin-Elmer, NEL103001EA, Waltham, USA). Three replicates were performed for each experiment.

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