Figure 5
BAG3_Pro209 mutants are trapped in long-lasting aggresome structures due to reduced subunit exchange. (a) Protein degradation rates were determined with a cycloheximide wash-out experiment. HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with wild type or mutant BAG3-GFP constructs and subjected to cycloheximide treatment (50 µg/ml) for the indicated time. Protein turnover of BAG3-GFP was determined by western blot after separation of the soluble from insoluble fraction. (n = 3). (b–d) Fluorescence recovery after photobleaching (FRAP) analysis was performed on HeLa cells that were transiently transfected with BAG3-GFP and mScarlet-Hsp70 or SQSTM1/p62-mCherry constructs. Bleaching was performed either on (b) BAG3-GFP, (c) mScarlet-Hsp70, or (d) SQSTM1/p62-mCherry. Quantification of the fluorescence intensity over time was plotted for cells overexpressing WT and mutant BAG3. Graph bar shows the means (±SD) over time (n = 6). Scale bar = 10 µm.
