Autophagy Inhibitor, 3-MA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
It interferes with LC3B-I-LC3B-II conversion and inhibits autophagosome formation.	Treat the cells for 1hour with 10mM 3-MA.

Upstream tips
It interferes with LC3B-I-LC3B-II conversion and inhibits autophagosome formation.
Protocol tips
Treat the cells for 1hour with 10mM 3-MA.

Publication protocol

RIPA lysis buffer (Biyuntian, Nanjing, China) was applied to achieve cell or tissue protein lysates. The protein concentration was determined by the Bio-Rad reagents (Bio-Rad, Shanghai, China). Aliquots of 30 μg protein lysates per condition were separated by 10-12% of SDS-PAGE gels, followed by transferring to the PVDF membranes (Millipore, Suzhou, China). Afterward blocking in 10% milk, the blots were probed with indicated primary and secondary antibodies. Afterward, the ECL reagents (GE, Shanghai, China) were applied to visualize the targeted protein bands. Band intensity was always quantified via the ImageJ software (NIH), and the value was normalized to the loading control [46].

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