DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative associated proteins (2016)
Figure 6

Figure 6. The Hsc70 chaperone is required for DnaJC5-mediated release.

A. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular tau levels as shown by dot blot. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

B. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular synuclein levels as shown by dot blot. Intracellular levels are shown by Western blot DnaJC5 was detected by FLAG antibody. Quantification of extracellular synuclein levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

C. Dominant-negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot. Intracellular levels are shown by Western blot. Quantification of extracellular tau levels is shown as average ± SEM, n=4, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

D. Knockdown of Hsc70 reduces extracellular tau release (dot blot) in HEK293T cells overexpressing FLAG-DnaJC5. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

E. A mutant tau that does not bind Hsc70 is not released when FLAG-DnaJC5 is overexpressed in HEK293T cells. Dot blot and quantification of extracellular tau levels are shown. Intracellular protein levels are shown with representative Western blot, DnaJC5 was detected by FLAG antibody. Data are mean ± SEM, n=3 p<0.001. one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

F. Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. HSPA variants were detected with V5 antibody. Data are mean ± SEM, n=3 p<0.01 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

G. Dot blot of different FLAG-DnaJ overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. DnaJ levels were detected by FLAG antibody. Data are mean ± SEM, n=3; *** p<0.001 one-way analysis of variance with Tukey’s multiple comparison post-hoc analysis.

H. Dot blot of increasing FLAG-DnaJC7 levels on FLAG-DnaJC5 overexpression-mediated extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot, DnaJ proteins were detected by FLAG antibody. Quantification of extracellular levels are mean ± SEM, n=4.

I. Schematic representation of Hsp70 control of DnaJC5-mediated tau release. When clients are unable to bind Hsc70, or if Hsc70 is inactive or knocked down, client proteins are sent for degradation instead of release. Thus, Hsc70 is important for upstream control of the DnaJC5-mediated extracellular release.