Identification of LMNB2 ubiquitinate sites by mass spec

HEK293T cells were transfected with Flag-LMNB2, HA-Ub, and MYC-SPOP plasmids. After 48 hours, the proteins were extracted using IP buffer supplemented with protease and phosphatase inhibitors. The lysates were incubated on ice for 10 minutes, followed by centrifugation at 13,000 rpm for 15 minutes at 4°C. Subsequently, 100 µL of Flag-conjugated agarose beads were added to the supernatant and incubated overnight at 4°C. The beads were washed at least four times with IP buffer, and proteins were eluted by adding 1.5× SDS buffer and heating at 100°C for 10 minutes. The supernatant was then collected and subjected to downstream Liquid Chromatography-Mass Spectrometry (LC-MS) analysis, a process technically supported by Novogene. Protein identification and quantification were performed using Proteome Discoverer (DDA) 2.5. Ubiquitination sites were identified as a mass shift of 114 Da on lysine residues, which corresponds to the di-glycine (GG) remnant left after tryptic digestion of ubiquitinated proteins. The identified ubiquitinated peptides were further manually inspected to confirm the correct peptide sequences and modification sites.