Fig. 2.

Protein aggregation by BAG3P209L is not caused by a loss of HSPB binding. a Binding of BAG3 to HSPB1c (Hsp27c) is partially disrupted by P209L. Binding affinity and estimated stoichiometry were measured by ITC. Results are the average of at least three independent experiments and error is standard deviation (SD). b Immunoprecipitation from HEK293 cells expressing FLAG-BAG3^WT or mutant variants using anti-FLAG beads. Western blots for FLAG (BAG3) and MYC (HSPB8) is shown. * indicates a cross-reactive band used as loading control. c NP-40 insoluble fraction of HEK293 cells expressing indicated FLAG-BAG3 variants. Western blot against the indicated antibodies is shown. The soluble fraction can be found in supplemental figure S2B. d Immunofluorescence pictures of HeLa cells expressing FLAG-BAG3^WT or indicated mutants, using a BAG3 antibody (green). Scale bar = 5 μm. e Quantification of the percentage of cells with BAG3 aggregates expressing the indicated variants of BAG3. Data represent the mean and standard deviation of two independent experiments (at least 100 cells were counted per experiment, Welch t-test was used to calculate the P values, * indicates P value < 0.05 and ns is not significant). Source data are provided as a Source data file