VCPIP1 Drives Diabetic Cardiomyopathy by Deubiquitinating AMPKγ1 and Preventing AMPKα-γ Subunit Assembly in Cardiomyocytes

Cardiomyocytes were transfected with either Flag-VCPIP1 or Flag-empty vector (EV) plasmid for ubiquitin proteomics analysis. In brief, cell samples were lysed ,and the supernatant was collected and protein concentration was determined. For each sample, the protein was reduced with dithiothreitol at 56°C and subsequently alkylated with iodoacetamide at room temperature in the dark. The proteins were first digested with trypsin at a trypsin-to-protein ratio overnight at 37°C, followed by a second digestion at 37°C. The resulting peptides were divided for ubiquitin peptide enrichment and protein quantification. For ubiquitin remnant enrichment, peptides were dissolved in NETN buffer and incubated with pre-washed antibody beads at 4°C overnight with gentle agitation.