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[Preprint]. 2024 Oct 13:2024.10.12.618026. [Version 1] doi: 10.1101/2024.10.12.618026

Figure 1: Pol η is a substrate of NEDD8.

Figure 1:

(A) Schematic of Pol η illustrating the location of lysine residues that can be mono-ubiquitinated, and putatively, mono-NEDDylated. (B) WT FLAG Pol η was immuoprecipitated from 293T cells and the lysate and eluent immunoblotted as indicated. FLAG and NEDD8 antibodies were detected with fluorescent secondary antibodies and visualized on separate channels. The top box shows a merged image of both channels. (C) WT and mutant FLAG Pol η was immunoprecipitated from 293T and the lysate and eluent immunoblotted as indicated. Relative NEDDylation was calculated based on the ratio of NEDD8 to FLAG in the eluent. The graph represents the quantification of relative NEDDylation from three independent repeats. Error bars represent standard deviation. Unpaired t-tests were used to assess differences in NEDDylation of WT vs mutant Pol η. ** = p < 0.1. (D) WT and 4KA FLAG Pol η was immunoprecipitated from 293T cells co-expressing HA NEDD8. The lysate and eluent were immunoblotted as indicated. Relative NEDDylation was calculated based on the ratio of HA to FLAG in the eluent. Relative UBLylation was calculated based on the ratio of higher to lower FLAG Pol η bands. The graph represents quantification from three independent repeats. Statistical analysis was performed as per (C). *** = p < 0.01.