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. Author manuscript; available in PMC: 2018 May 4.
Published in final edited form as: Cell. 2017 May 4;169(4):722–735.e9. doi: 10.1016/j.cell.2017.04.020

Figure 6. Ubiquitin passes through the central pore of Cdc48.

Figure 6

(A) SBP-tagged Cdc48 (Cdc48-SBP) with Bpa at the M288 position (D1 pore loop) was incubated with dye-labeled free, K48-linked polyubiquitin chains and the indicated combinations of UN and nucleotide. After irradiation and streptavidin pulldown, crosslinked species were detected by SDS-PAGE and fluorescence scanning. The silver-stained band of Cdc48-SBP serves as a loading control.

(B) As in (A), but with Bpa at the D324 position in the interior of the central pore.

(C) As in (A), but with Bpa at the D602 position in the D2 pore-2 loop. Brackets, non-crosslinked material.

(D) As in (A), but with Bpa at positions M288 or D324 and either penta-ubiquitin ((Ub(5)) or polyubiquitin (Ub(n)). The arrowhead indicates a weak crosslink of Ub(5) to position 288. M, molecular weight markers.