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. Author manuscript; available in PMC: 2015 Nov 6.
Published in final edited form as: Mol Cell. 2014 Oct 2;56(3):360–375. doi: 10.1016/j.molcel.2014.09.007

Figure 4. UB chain synthesis by PINK1-phosphorylated PARKIN in vitro.

Figure 4

(A) Schematic for preparation of PINK1Tc-phosphorylated PARKIN free of PINK1Tc.

(B) The stoichiometry of PARKIN phosphorylation at S65 was determined by AQUA proteomics.

(C) UB chain synthesis by WT, S65A, or C431S PARKIN with or without phosphorylation by PINK1Tc followed by removal of PINK1Tc was measured by immunoblotting with α-UB antibodies. Control blots were probed with α-PARKIN or α-p-S65 (AVI-25) (see Extended Experimental Procedures).

(D,E) UB-AQUA of total UB linkages (Kgg) (D) and individual linkage types (E) for reaction mixtures (45 min) from the indicated PARKIN proteins.

(F) UB chain editing of PARKIN ubiquitylation products assembled in vitro. DUBs were incubated with UB chains assembled by p-WT PARKIN and the products analyzed by immunoblotting. As controls, linear or K48 UB chains were digested with the indicated DUBs.

See also Figure S4.