Figure 5.

IAPs do not target cleaved caspsase-3 for ubiquitin-proteasome-mediated degradation in NGF-treated cells. PC12 cells were treated with NGF for the indicated times, and changes in the levels of IAPs were analysed. (a) Semi-quantitative reverse transcriptase-PCR was performed to determine XIAP, IAP1 and IAP2 mRNA levels. GAPDH was used as a house-keeping control. (b) Western blotting was used to visualise XIAP and cIAP1/2 protein levels. Actin was used as a loading control. (c) PC12 cells were treated with 1.5 μM TG for 18 h before NGF addition. The cells were harvested at 6 h after NGF addition, and the protein levels of XIAP were determined using western blotting. Actin was used as a loading control. (d) PC12 cells were treated with 5 μM BV6 for the indicated times, and western blotting was performed using anti-IAP1/2 antibody. (e) PC12 cells were treated with 1.5 μM TG for 14 h followed by addition of 5 μM BV6. At 20 h, NGF was added for the indicated times. The cells were harvested for western blotting analysis at 22–26 h as indicated. The p17 subunit levels were analysed using the cleaved caspase-3 antibody. Actin was used as a loading control. Results shown are representative of three distinct experiments. (f) PC12 cells were treated with 10 or 20 μM MG132 for the indicated times. The cells were then harvested, and the total levels of ubiquitinated proteins were analysed using an anti-ubiquitin antibody. (g and h) PC12 cells were treated with 1.5 μM TG for 19 h followed by incubation with 20 μM MG132 (g) or 10 μM Ub-EI (h) for 1 h before addition of NGF. Cells were harvested at 20–26 h as indicated. The p17 subunit levels were analysed using the cleaved caspase-3 antibody. Actin was used as a loading control. Results shown are representative of three distinct experiments