300,000 cells were seeded and transduced with MKRN3 and control lentivirus. 60 h later, the medium was replaced with methionine-free medium (Thermo Fisher Scientific; #A1451701) for 2 h. Cells were incubated with 50 µM L-AHA (Clickchemistrytools; #1066-25) for 8 h and harvested in lysis buffer. L-AHA was detected using click chemistry with 50 µM Biotin Alkyne (Clickchemistrytools; #1266–5) with the Click-&-Go Protein Reaction Buffer Kit (Clickchemistrytools; #1262) per the manufacturer’s protocol. The extracted protein was detected by Western blotting as described above. The blots were probed with a horseradish peroxidase–conjugated streptavidin (Proteintech; #SA0001) and developed with Enhanced Chemiluminescence (GE; #RPN2232).
Li K., Zheng X., Tang H., Zang Y.S., Zeng C., Liu X., Shen Y., Pang Y., Wang S., Xie F., Lu X., Luo Y., Li Z., Bi W., Jia X., Huang T., Wei R., Huang K., Chen Z., Zhu Q., He Y., Zhang M., Gu Z., Xiao Y., Zhang X., Fletcher J.A, & Wang Y. (2021). E3 ligase MKRN3 is a tumor suppressor regulating PABPC1 ubiquitination in non–small cell lung cancer. The Journal of Experimental Medicine, 218(8), e20210151.
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