Fig. 4: Validation of the substrate candidates for Parkin and TRIM28 E3 ligases. | Communications Biology

Fig. 4: Validation of the substrate candidates for Parkin and TRIM28 E3 ligases.

From: A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets

Fig. 4

a, b Schematic presentations indicating the steps in the ubiquitination assay using recombinant GST–FLAG–TUBE (a) or FLAG–TUBE proteins (b). c HEK293T cells stably expressing HA–Parkin or harboring an empty vector (Mock) were not treated with or were treated with CCCP (10 μM) for 1 h. Cell lysates were pulled down with the beads prebound by GST–FLAG–TUBE proteins. The precipitates were analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated substrates and unmodified substrates, respectively. d HEK293T cells with stable knockdown of TRIM28 or corresponding control cells were harvested. Cell lysates were pulled down with the beads prebound by FLAG–TUBE proteins. The precipitates were analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated substrates and unmodified substrates, respectively.

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