High capacity streptavidin magnetic beads

MDA-MB-231 and 4T1 cells were cultured with glucose/glutamine-deficient medium or supplemented with inosine or glucose for 8 h, then added 20 μM OPP (Cat#1407-5, Click Chemistry Tools) to the medium for further culturing 2 h. Subsequently, cells were collected to extract the proteins on ice. Totally, 250 µg proteins were combined with biotin by click chemistry, allowing biotin picolyl azide (Cat#1167-5, Click Chemistry Tools) and Click-&-Go® Protein Reaction Buffer Kit (Cat#1262, Click Chemistry Tools) from Click Chemistry Tools following the provided instructions. After that, methanol and chloroform were added to the mixture to precipitate proteins, then washed with methanol. The pellets were resuspended in PBS with 1% SDS and measured protein concentration by BCA assay after air drying. Ten micrograms of protein were stored at −80 °C as input control. Totally, 150 µg of proteins were incubated with High Capacity Streptavidin Magnetic Beads (Cat#1497-1, Click Chemistry Tools) at 4 °C overnight with slow rotation. Global nascent proteins were dissolution with 2× loading buffer in the supernatant fluid after beads elution. The proteins were prepared for LC/MS analysis by SDS-PAGE electrophoresis. The nascent proteins are shown in Supplementary Table 3.

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.