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. 2023 Apr 15;14:2160. doi: 10.1038/s41467-023-37504-x

Fig. 5. Substrate ubiquitination parallels substrate degradation during Trim-Away in cells.

Fig. 5

a Schematic showing Trim-Away experiment in which antibodies are electroporated into cells expressing endogenous TRIM21. Ubiquitination and degradation are then monitored in the presence or absence of proteasome inhibitor MG132. b, c RPE-1 cells were electroporated with PBS or anti-ERK1 antibody (b) or anti-IKKα antibody (c) and whole cell lysates harvested at the indicated times after electroporation for immunoblotting. Short exposures show degradation of substrates. Long exposures reveal substrate ubiquitination followed by degradation of ubiquitinated species. d RPE-1 cells were electroporated with PBS or anti-IKKα antibody ± MG132 and whole cell lysates harvested 1 h post-electroporation for immunoblotting. e RPE-1 TRIM21 KO cells reconstituted with TRIM21-HA (T21-HA) or empty vector (EV) were electroporated with PBS or anti-ERK1 antibody ± MG132 and whole cell lysates harvested 1 h post-electroporation for immunoblotting. Representative examples (b–e) from n = 3 independent experiments. Source data are provided as a Source Data file. See also Supplementary Fig. 5.