Figure 4 | Cell Death & Disease

Figure 4

From: P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase complex HSP70/CHIP

Figure 4

The p42 promotes p85 degradation through ubiquitin–proteasome system. (a) GFP-p42 (1 and 3 μg) was transfected into HEK293 cells, following exposure to 10 μM MG132 for 8 h. Immunoblotting was conducted to monitor p85, p42 or β-actin levels. (b) PC12 cells were co-transfected with HA-Ub and Flag-p85 and/or GFP-p42/p48 for 30 h and treated with MG132 (10 μM) for an additional 8 h. To obtain similar amount of immunoprecipitated Flag-p85, we transfected 0.5 μg of GFP-p42 while we transfected 1 μg of GFP or GFP-p48. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA antibody. (c) HEK 293T cells were co-transfected with Flag-p85 and HA-Ub along with N-si-p48 or si-Ebp1 following exposure of MG132. Ubiquitinated p85 was detected by immunoblotting with anti-HA antibody. (d) Cells were transfected with various Flag-p85 fragments with or without GFP-p42, and p85 levels were determined by anti-Flag antibody (left). Densitometry analysis (right). **P<0.005

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