Figure 7.

Rapid Antibody RING-Mediated Destruction of Endogenous NEDP1

(A, D, and G) Nickel bead pull-down assays of recombinant 6His-NEDP1 with nanobody-RING fusions NNb2-1xRING and 2xRING (A), NNb9-1xRING and 2xRING (D), and NNb7-1xRING and 2xRING (G) were evaluated with SDS-PAGE and Coomassie staining (In, input; S, supernatant; P, pull-down). Fused RNF4 RING (2xRING) is used as negative control.

(B, E, and H) The activity of the NNb2-1xRING and 2xRING (B), NNb9-1xRING and 2xRING (E), and NNb7-1xRING and 2xRING (H) was tested in lysine discharge assays with ubiquitin loaded Ubc5 (Ub-Ubc5). A fused RNF4 RING (2xRING) served as a positive control. Samples were removed at indicated times (minutes) and analyzed by non-reducing SDS-PAGE.

(C, F, and I) To assess activity of purified recombinant NEDP1 ARMeD fusions in cells, HEK293 cells were electroporated with NNb2-1xRING and 2xRING (C), NNb9-1xRING and 2xRING (F), or NNb7-1xRING and 2xRING (I) and harvested at the indicated time point after electroporation. NEDP1 and NEDD8 were analyzed by western blotting. NEDP1, a non-specific band (NS), NEDD8-cullins, and NEDD8 monomers and dimers are indicated by arrows.

See also Figures S5 and S6.