Figure 4
BAG3_Pro209 mutants sequester other members of the CASA-complex in aggresomes. HEK293T cells that stably overexpress HSPB8-V5 were transiently transfected with wild type or mutant BAG3-GFP constructs to assess the interaction between BAG3 and components of the CASA-complex. (a) Co-immunoprecipitation of BAG3-GFP and the CASA-complex using the GFP-trap system. The amount of interacting proteins was quantified and corrected for the amount of immunoprecipitated BAG3 as represented in the graph bar (means ± SD). One-Way ANOVA with Bonferroni’s multiple comparisons test were used for statistical analysis. NS = non-significant, **p < 0.01, and ****p < 0.0001 (n = 3). The wild type (WT) or mutants were abbreviated as followed: Pro209Ser (PS), Pro209Leu (PL), Pro209Gln (PQ), Glu455Lys (EK). (b–d) Immunocytochemistry of BAG3-GFP constructs to assess colocalization with (b) endogenous Hsp70, (c) HSPB8, and (d) SQSTM1/p62. Scale bar = 5 µm (b) and 10 µm (c,d). (e,f) Live-cell time-lapse imaging of GFP-tagged BAG3_Pro209Leu and RFP-tagged SQSTM1/p62 or Hsp70. HeLa cells were transiently transfected with mutant BAG3-GFP constructs and (e) mCherry-tagged SQSTM1/p62 or (f) mScarlet-tagged Hsp70. Cells were imaged once per hour. Scale bar = 50 µm.
