Fig. 1: Development of PBSP to track the spatiotemporal secretion of itaconated proteins in living macrophages. | Nature Communications

Fig. 1: Development of PBSP to track the spatiotemporal secretion of itaconated proteins in living macrophages.

From: Spatiotemporal profiling of modification-specific proteome secretion uncovers an itaconation-activated tyrosine kinase

Fig. 1

a Schematic illustrating the pulse-chase labeling of itaconated secretomes with ITalk. Macrophages were pulse-labeled with ITalk for 2 hours, followed by probe washout and a 12 hours chase period in serum-free medium. Secreted proteins in the medium were then collected for click reaction-based detection. Created in BioRender. Chu, l. (2025) https://BioRender.com/p66bt7z. b Effect of the PBSP workflow on itaconate levels. Metabolites were extracted from Raw264.7 cells after the complete pulse-chase procedure and itaconate was quantified by LC-MS/MS. For the positive control, cells were stimulated with LPS (100 ng/mL) for 14 hours. Quantification was performed from three biological replicates and the error bars show mean ± SD. ***p < 0.001 (two-sided Student’s t-test). p-values: 0.11317 (2 hours), 0.41508 (14 hours), 0.00001 (Lps 12 hours). c In-gel fluorescence detection of ITalk labeling in the culture medium (left) or cell lysates (right). Raw264.7 cells were treated with 0, 1, or 5 mM ITalk for 2 hours, followed by a 12 hours chase in serum-free medium. d Streptavidin blot (SA-blot) detection of distinct probe labeling in the culture medium (left) or cell lysates (right). For (c, d), both the culture medium and cells were subjected to protein extraction and click reaction with azide-Rhodamine or azide-biotin. Labeling intensity was determined by in-gel fluorescence scanning or SA-blot, and Coomassie Brilliant Blue (CBB) staining was used to confirm equal protein loading. Three replicates were performed with similar results. Corresponding uncropped images are shown in the Source Data file.

Back to article page