Were transfected with INrf2 or Cul3 siRNA Bcl2 and ubiquitination was analyzed. The results showed that the inhibition of INrf2 or Cul3 siRNA resulted in a significant decrease in the ubiquitination NVP-LDE225 LDE225 and increased Hte stabilizing Bcl 2 is passed. In addition, overexpression of Myc RBX1, Cul3 H magglutinin Flag or INrf2, individually or combined degraded cellular Bcl 2 Ren by erh Hte ubiquitination of Bcl second Interestingly, the overexpression of INrf2 had a gr Ere St Strength of the effect, probably. Because of its function as an adapter between 2 and Bcl Cul3 RBX1 Analysis of the mouse Bcl 2 amino Acid sequence revealed the presence of only four lysine residues at the amino Urepositionen 17, 22, 214 and 236 have proved to be U Conserved only in humans and rats.
Mouse Bcl 2k17, K22, K214, K236 and individually mutated to alanine in order to identify the target lysine Cilomilast INrf2-mediated ubiquitination. Wild type and mutant Bcl 2 were analyzed for endogenous INrf2-mediated degradation and ubiquitination in Hepa cells 1. Only the mutant Bcl2K17A, and not other mutants showed a significant reduction stabilization and ubiquitination. The INrf2 mediated ubiquitination specific wild type Bcl 2 and Bcl investigate two lysine mutants, we used 293 cells and INrf2 293 cells. The cells were transfected with Bcl V5 or lysine mutants 2 and HA ubiquitin for 24 h co-transfected. Subsequently End an amount of cells with tetracycline for 12 h and analyzed 2 and Bcl ubiquitination treated. The results show that overexpression of Bcl INrf2 flag greatly increased 2 ubiquitination Ht.
Zus Tzlich Bcl 2K17A mutant showed a significant decrease in ubiquitination INrf2 293 cells. These data also suggested that K17 Bcl 2 is the capital of the INrf2: Cul3 RBX1 induced ubiquitination of Bcl second This conclusion was supported by in vitro studies. Hepa 1 cell extracts overexpressing INrf2: Cul3 transcribed RBX1 protein ubiquitin and degraded in vitro / translated wild-type and lysine mutants K22A Bcl2 V5 V5 V5 V5 and K236A K214A, but not mutant Bcl 2K17A V5. Hepa 1 extract overexpressing INrf2: Cul3 RBX1 also showed ubiquitination and degradation of purified bacterial Bcl second INrf2 through its DGR Dom ne interacts with the BH2 Dom ne second of Bcl INrf2-mediated ubiquitination / degradation of Bcl-2 and Bcl 2 regulation of stability T suggested that INrf2 and Bcl 2 interact.
Experiments examined endogenous INrf2 interaction and Bcl 2 in Hepa 1 cells, the interaction of tetracycline induced INrf2 flag with endogenous Bcl 2 in 293 cells and INrf2 INrf2 interaction with Bcl 2 flag V5 INrf2 transfected 293 cells. Anti INrf2 antique Body immuno INrf2 and Bcl 2 was pulled down with him. In reverse Immunpr Zipitation, anti-Bcl 2-antique Body immuno INrf2 and Bcl second These results showed INrf2 second interaction with endogenous Bcl Moreover, pulling Immunpr Zipitation the flag down INrf2 endogenous Bcl 2 and vice versa INrf2 in 293 cells, but not in the embroidered the 293 cells. Moreover, pulled Immunpr Zipitation the flag INrf2 Bcl 2 V5 INrf2 HEK 293 cells with Bcl 2 cells transfected V5 and vice versa. As n We chstes interaction Dom NEN INrf2 2 and Bcl-proteins Mapped. INrf2 has identified five different areas