Supplementary Figure 7: Ub phosphorylation at Ser65.
From: Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30
(a) Schematic drawings of phosphorylated Lys6-Ub2 preparation. pSer65-containing Ub (produced by Tribolium castaneum PINK1) and non-phosphorylated (native) Ub were enzymatically linked by E1, NleL and UbcH7. Lys48 of all the Ub molecules was mutated to Arg to avoid elongation of the Lys48-linked chains. For efficient production of Ub2, Lys6 of Ubdist was mutated to Arg, and Asp was attached to the C-terminus of Ubprox. See also online methods. (b) SDS-PAGE of the purified Ub and Lys6-Ub2 with or without phosphorylation. The left panel shows the result in the absence of Phos-tag, whereas the right panel shows that in the presence of Phos-tag. These data guarantee the purities of the phosphorylated samples.
