Figure 1
BAG3_Pro209 mutations cause cytoplasmic aggregation. (a) Schematic representation of the structure of BAG3, including the WW-domain, the two IPV-motifs, the PxxP-domain and BAG-domain. The known interactors of each motif are shown at the top and the missense mutations that were studied in this manuscript are shown at the bottom in red. (b) HEK293T cells stably expressing HSPB8-V5 were transiently transfected with BAG3-GFP constructs. Six random fields were selected for analysis. The mean number of cells counted per field was 95 and thus over 400 cells per genotype were counted. (scale bar = 10 μm) (c) Quantification of BAG3-GFP inclusions using Flow cytometric analysis of inclusions (FloIT). Transiently transfected HEK293T cells were collected and stained with DAPI prior to 0.1% Triton X-100 treatment. The intracellular BAG3-GFP inclusions and Hoechst-positive nuclei are subsequently quantified using flow cytometry. Bar graph represents the means of BAG3-GFP cytoplasmic inclusions per 100 transfected cells. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis. (d,e) Bio-informatic analysis of (d) the solubility of wild type or mutant BAG3 with CamSol and (e) of the aggregation propensity with Tango software. (f) Western blot analysis of the NP-40 soluble fraction from HEK293T cells stably expressing HSPB8-V5 and transiently transfected with BAG3-GFP constructs. The constructs were abbreviated as followed: wild type (WT), Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK). One of three representative western blots is shown. (g) Filter retardation assay (FRA) analysis of the NP-40 insoluble fraction. Anti-GFP and anti-HSPB8 antibodies were used to detect insoluble levels of BAG3 (wild type or mutants) and HSPB8. Relative optical densities are reported in the graphs as means ± SD of normalized values. One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis (n = 3). The constructs were abbreviated as followed: non-transfected (NT), empty vector (EV), wild type (WT), Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK).
