Fig. 6
From: Peli1 negatively regulates noncanonical NF-κB signaling to restrain systemic lupus erythematosus
Peli1 binds to and mediates Lys48 ubiquitination of NIK. a Immunoblot analysis of NIK and HSP60 in whole-cell lysates of WT and Peli1-deficient splenic B cells stimulated with anti-CD40 (αCD40). b Analysis of Lys48 ubiquitination of NIK in WT and KO B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of a proteasome inhibitor MG132. IP, immunoprecipitation; IB, immunoblotting. c Ubiquitination of NIK in HEK293 cells transfected with (+) or without (−) indicated expression vectors. d Immunoassays on lysates of WT splenic B cells left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132, followed by IP with control IgG or anti-NIK and immunoblot analysis of NIK-associated Peli1. e IP analysis examining the association of NIK with Peli1 in HEK293 cells transfected with (+) or without (−) indicated expression vectors. f Immunoblot analysis of Lys48 ubiquitination of NIK assessed by in vitro ubiquitination assay with a mixture of E1, E2 (UbcH5c), ATP, Flag-NIK, HA-Peli1, or HA-Peli1ΔC after IP with anti-Flag. g Immunoblot analysis of p52, p100, NIK, c-IAP2, Peli1, and HSP60 in total lysis of control and Peli1-knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, and then stimulated with anti-CD40 (αCD40) (upper panel) or BAFF (lower panel) at the indicated time points. Ctrl, control. h Analysis of Lys48 ubiquitination of NIK in control and Peli1-knockdown M12 cells that pretreated with DMSO or smac mimetic BV6 for 4 h, then left unstimulated or stimulated with anti-CD40 (αCD40) for 4 h in the presence of MG132. Ctrl, control. Data are shown based on three independent experiments
