Fig. 2: PML lysine 394 is required for maintaining USP22-dependent PML stability.

A Schematic overview of PML mRNA (blue) with approximate exon spans indicated, as well as the PML (black) and PML-RARα protein (yellow) with relevant amino acids indicated (K, lysine; A, alanine; I, isoleucine; S, serine). RBCC/Trim motif, RING-B-box1-B-box2-Coiled-Coil domain; NLS, nuclear localization signal; SIM, SUMO-interacting motif. B Western blot analysis of HEK293T cells transiently transfected with plasmids expressing wild-type (WT) and K394R HA-tagged PML isoform IV. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. C Western blot analysis of HEK293T cells transiently transfected with plasmids expressing WT and K394R HA-PML isoform IV, prior to treatment with 20 μg/ml CHX for the indicated timepoints. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. D Densitometric quantification of gray level intensities of WT and K394R HA-PML isoform IV detected by Western blot analysis of HA in HEK293T cells in the presence of CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent biological replicates are shown. E Western blot analysis of anti-ubiquitin (Ub), -HA and -PML on anti-HA-immunoprecipitated fractions of denatured lysates of HEK293T cells transiently transfected with empty vector (EV) or plasmids expressing WT and K394R HA-PML isoform IV. ß-Actin served as loading control. Representative blots of at least two different independent experiments are shown.