Fig. 5.

BAG3^P209L aggregation requires an interaction with Hsp70. a Immunofluorescence images of HeLa cells expressing FLAG-BAG3^wt and indicated mutants using an antibody recognizing BAG3 (green). Scale bar = 5 μm. b Quantification of the fraction of HeLa cells expressing the indicated BAG3 variants with immunofluorescence detectable punctae (2 experiments, at least 100 cells were counted per experiment, error bars represent SD, Welch t test was used to calculate the P values, * indicates P < 0.05). c Western blot of the NP-40 insoluble fraction of HEK293 cells expressing BAG3^wt and indicated variants of BAG3. d Immunoprecipitation of FLAG-BAG3 variants from HEK293 cells expressing both Myc-HSPB8 and indicated FLAG-BAG3 variants. Western blots using the indicated antibodies is shown. e Western blot of NP-40 soluble and insoluble fractions of HEK293 cells expressing indicated BAG3 variants; FLAG (BAG3), Myc (HSPB8), and tubulin antibodies were used. f Western blot of immunoprecipitates using anti FLAG beads from HEK293 cells expressing both FLAG-BAG3^P209L and Myc-HSPB8 in cells treated with either 0.5 μM JG-98, 5μM YM-01 or DMSO; FLAG (BAG3), Myc (HSPB8), HSPA1A, and actin antibodies were used. g, h Western blot of NP-40 soluble and insoluble fractions of HEK293 cells expressing both FLAG-BAG3^P209L and Myc-HSPB8, treated with DMSO or increasing concentration of the drug JG-98 (0.25, 0.5, or 1.0 µM) (g) or YM-01 (1.25, 2.5, 5.0, or 10 µM) (h). Source data are provided as a Source data file