Figure 5

The p42 recruits a chaperone-E3 ligase complex HSP70/CHIP for p85 degradation. (a) HEK293T cells were transfected with various GFP-Ebp1 fragments, and p85 levels were examined by immunoblotting. (b) p42 interacts with HSP70 and CHIP. 293T cells were transfected with GST-Ebp1 (280–394 a.a.). After GST pull-down assay, the samples were subjected to silver staining according to the manufacturer’s instructions and applied for mass-spectrometry. (c) GST-pull down analysis was performed and immunoblotted with the indicated antibodies (left). 293T cell lysates were immunoprecipitated with anti-HSP70 antibody (upper) or anti-CHIP antibody (bottom) and immunoblotting with anti-Ebp1 or anti-N-Ebp1 antibody (right) (d) knockdown experiment with si-HSP70. (e and f) Ubiquitinated p85 was detected by immunoblotting with anti-HA antibody from Flag-p85- and HA-Ub-transfected PC12 cells. To obtain similar amount of immunoprecipitated Flag-p85, we transfected 0.5 μg of GFP-p42 while we transfected 1 μg of control. (g) p85 levels were monitored after co-transfection with different combinations of Myc-p42, GST-CHIP or HSP70. (h) Endogenous p85 levels were determined from 293T cells silenced with SCR, si-Ebp1 or si-CHIP (upper). Densitometry analysis (bottom). **P<0.005 versus control. (i) Endogenous protein levels were examined in the mouse embryonic fibroblast (MEF) CHIP ^(−/−) or CHIP ^(+/+) cells against anti-p85, anti-Ebp, anti-CHIP or anti-β-actin antibodies. (j) MEF CHIP ^(−/−) or CHIP ^(+/+) cells were co-transfected with Flag-p85 and HA-ub along with GFP-p42 and then exposed to MG132 for 8 h. (k) CHIP ^(−/−) cells was transfected with Myc-CHIP with or without p42 and immunoblotting was performed as indicated. (l) PI3K activity assay with GFP-p42- or Myc-CHIP-transfected cell lysates. *P<0.05 and **P<0.005