Fig. 9. Two ubiquitin ligases are involved in p105 processing, one of them, SCF^β-TrCP, requires the presence of residues 918–934 and, in particular, serine residues 922, 924 and 933. (A) SCF^β-TrCP conjugates wild-type and Δ446–454, but not Δ918–934 and Δ446–454-S922,924,933A p105s. In vitro translated and kinase-phosphorylated wild-type (lanes 1–3), Δ446–454 (lanes 4–6), Δ918–934 (lanes 7–9) and Δ446–454-S922,924,933A (lanes 10–12) p105s were subjected to in vitro conjugation in a reconstituted cell-free system as described in Materials and methods. When indicated, Ubc5Hc and SCF^h-β-TrCP were added as described. (B) Conjugation of wild-type and Δ918–934 p105s in crude HeLa extract. In vitro translated and kinase-phosphorylated wild-type (lanes 1 and 2) and Δ918–934 (lanes 3 and 4) p105s were subjected to in vitro conjugation as described in Materials and methods. The cell-free system contained crude HeLa cell extract as a source of the conjugating enzymes. Proteins were resolved and visualized as described in Materials and methods.