FIGURE 8.

Recombinant Hsc70 is capable of restoring TH levels at the synaptic vesicle in Hsc70-shRNA cells. The synaptic vesicle fractionations were repeated under conditions where S2 was incubated alone (Control) or in combination with either recombinant Hsp70 or Hsc70 prior to obtaining S3 and P3. 50 μg of total proteins from H, S3, and P3 of WT, scramble shRNA, and Hsc70 shRNA MN9D cells were analyzed by SDS-PAGE and IB. Again, TH was enriched in P3 of WT and scramble shRNA cells in S3 of the Hsc70 shRNA cells under normal conditions. Importantly, incubation with Hsc70 restored TH levels to the P3 fraction in Hsc70 shRNA cells. Incubation with the closely related Hsp70 protein had no effect on TH distribution, confirming the specificity of the Hsc70 effect. IB analysis with SYPH, VMAT2, SV2, and secretogranin II antibodies confirmed that the fraction P3 contained synaptic vesicles.