Newly Translated Protein Synthesis Assay

A newly translated protein assessment was performed according to the previous investigation^{43 (link)}. Briefly, BMDMs that were treated with or without 100 ng/ml LPS for 6 h were considered as 0 h samples. 3.5 or 4.5 h after treatment, the culture media of BMDMs were changed into methionine (Met)-free RPMI1640. 30 min later, the media were replaced with Met-free RPMI1640 including AHA (40 mM), and incubated for one hour (1 h samples) or two hours (2 h samples), respectively. After 6 h, BMDMs were lysed in 50 mM Tris-HCl (pH 8.0) with protease inhibitors and 1% SDS by sonication and incubated on ice for 30 min. Cell lysates were centrifuged at 12,000 × g for 20 min and the supernatants were harvested. The Click & Go Protein Reaction Buffer Kit (Click Chemistry Tools, Cat#: 1262) and Biotin-PGE4-Alkyne were used to treat cell lysates for biotinylation. 10% of the recovered proteins were saved as input controls, and the other 90% of proteins were subjected to streptavidin-coated magnetic beads (High Capacity Streptavidin Magnetic Beads, Click Chemistry Tools, Cat#:1497-1) to enrich the AHA-labeled proteins. Both input and precipitated proteins were lysed in RIPA buffer, followed by western blot analysis.