Figure 8.

Chaperone-mediated autophagy is not involved in NGF-induced removal of p17. (a and b) PC12 cells stably transduced with shRNA to Hsc70 (a) or LAMP2a (b) were treated with 1 μM TG for 20 h followed by addition of NGF for further 2, 4 and 6 h. The p17 subunit levels were analysed by western blotting using the cleaved caspase-3 antibody. Actin was used as a loading control. Results shown are representative of three distinct experiments. (c) PC12 cells were treated with 1 μM TG for 19 h followed by treatment with 25 μM CQ for 1 h before addition of NGF. The cells were then harvested, and the lysate was incubated with cleaved caspase-3 antibody conjugated to Dynal Beads overnight. The beads were then applied to a magnet and washed. The protein was then eluted from the column and the lysate and the immunoprecipitated proteins were analysed by western blotting for p17 (using the cleaved caspase-3 antibody) and Hsc70. The data are representative of three separate experiments