In vitro ubiquitination assays. (A) Recombinant PI3K p110α was incubated with GST alone or GST-SOCS5-SH2-SOCS box in complex with Elongin B/C (GST-SOCS5-B/C), together with Cullin 5, Rbx2, ubiquitin and E2 enzyme at 37°C. Reactions were initiated by the addition of E1 enzyme and samples collected at the indicated times, prior to immunoblot analysis with anti-PI3K antibodies. The lack of higher molecular weight species in the presence of SOCS5 indicates that no SOCS5-mediated ubiquitination of PI3K p110α was observed. Exp: exposure. (B) Coomassie stained gel showing all components of the ubiquitination reaction. CTD: C-terminal domain, NTD: N-terminal domain, EloB: Elongin B, EloC: Elongin C. (C) Flag-tagged, full-length SOCS5 (F-SOCS5) was enriched from 293T cells and incubated at 37°C for 1 h with the components of the E3 ligase complex in the presence or absence of recombinant PI3K p110α. Immunoblot analysis of PI3K p110α indicates no SOCS5-mediated ubiquitination of PI3K (WT: wild-type; mSB: SOCS5-L484P, C488F, a non-functional SOCS box). (D) Immunoblot analysis with anti-Flag antibodies showing expression of SOCS5 prior to enrichment.