3.

Pulse-chase experiment to determine the dynamic nature of lysine malonylation. Wild-type mouse primary chondrocytes (A) and Sirt5 KD primary chondrocytes (B) were labeled with 200 μM MA-diyne for 1 h and then pulse chased with 200 μM Meldrum acid (precursor). The lysates were collected at the indicated time points followed by a click reaction with IR680-azide and in gel fluorescence analysis. β actin was used as a loading control. The same blot was probed with an anti-Sirt5 antibody to quantify the knockdown efficiency.