4.

Validation of lysine malonylated protein targets of MA-diyne. (A) Pull down of known malonylated proteins such as GAPDH and Aldolase A after enrichment using avidin beads. n = 2. (B) Pull down of newly identified proteins after MA-diyne enrichment. n = 2. (C) Pull-down of MA-diyne labeled GAPDH protein after overexpressing GAPDH wild type and GAPDH K184E plasmids in primary chondrocytes. Primary chondrocytes were overexpressed with GAPDH wild type and GAPDH K184E plasmids for 48 h and then labeled with MA-diyne. The protein lysates were clicked with biotin azide, and avidin beads were used to enrich the biotinylated proteins. The elute proteins from the avidin beads were resolved on SDS Gel and then transferred on a PVDF membrane. The blot was probed for anti-GAPDH antibodies. (D) Pie chart depicting the percentage of malonylated proteins found in different subcellular compartments by GO analysis. (E) Distribution of enriched proteins based on their biological process by GO analysis, (F) KEGG pathway analysis of the enriched proteins. p value < 0.05.