Fig. 2: Identification of E2 interactors in HEK293T cells via immunoprecipitation and mass spectrometry.
From: An adaptive stress response that confers cellular resilience to decreased ubiquitination
a An example of IP-MS of FLAG-tagged E2s to identify E2 interactors. Western blots for FLAG-tagged UBE2A detect the E2 bait, UBE2A, in input cellular extracts and following immunoprecipitation (IP) with anti-FLAG antibodies. Whole lanes of FLAG-IP for each E2 bait were excised and used for MS. b MS spectral counts were analyzed by SAINT to determine interacting proteins that were significantly enriched (SAINT score>0.65), which included E3s and deubiquitinating enzymes (DUBs). c Non-curated interactome of all proteins significantly interacting with the E1 and E2 baits (SAINT > 0.65); n = 3 biological replicates for each bait and n = 10 biological replicates for each control (no-FLAG baits). d Heatmap demonstrating that the FLAG-tagged E2 baits were detected and significantly enriched in their respective IP-MS. Cross-interactions were detected between E2s, even in cases where sequence homology is low (e). f Non-curated interactome of all proteins significantly interacting with E1 and E2 baits (SAINT > 0.65). The complex network integrates E1s for ubiquitin and ubiquitin-like proteins (UBLs), E2s, E3s, DUBs, proteasome and autophagy (ATGs) components, and other proteins. The UPS network of E1-E2-E3s interacts with the analogous E1-E2 autophagy network (ATG7, ATG3, and ATG10). Additionally, E2s such as UBE2C interact with proteasome components. g Curated interactome focused on UBLs, E1s, E2s, E3s and DUBs. This network, which is centered around ubiquitin (UB) and the UBA1 E1 enzyme, demonstrates specific UBL utilization, E1 and E2 cross-interactions, E2s with the same E3/DUB interactors and, conversely, with unique E3/DUB interactors. Source data are provided in the Source data file.
