Figure 3

The p42 regulates p85 protein stability. (a) HEK293T cells were co-transfected with GST-p110 (2 μg) and Flag-p85 (2 μg) along with different concentrations of Myc-p42 (2 or 4 μg). The cell lysates (1 mg) were subjected to a GST pull-down assay, and the interaction between p85 and p110 was shown by immunoblotting with anti-Flag antibody. Protein expression was evaluated by immunoblotting. (b) Cells from the breast cancer cell line MCF7 were transfected with GFP-p85 (2 μg) and RED-plasma membrane marker (1.5 μg) together with control (2 μg), Myc-p42 (2 μg) or Myc-p48 (2 μg). The serum-starved MCF7 cells were incubated with or without EGF (50 ng/ml) for 5 min before processing for immunocytochemistry. Cells immunostained with anti-Myc were immunoreacted with Alexa Fluor 350 donkey anti-mouse secondary antibody and observed using a Zeiss confocal laser-scanning microscope. p48 or p42 (gray), plasma membrane (red) and p85 (green). Scale bar: 10 μm. (c) Cells were transfected with Flag-p85 along with control or GFP-p42 (2 or 4 μg). The serum-starved cells were treated with EGF (50 ng/ml) for 5 min. The cell lysates were subjected to subcellular fractionation for membrane and cytosolic fraction. p85 protein levels were evaluated by immunoblotting with anti-Flag antibody. The purity of each fraction was examined by anti-tubulin (cytosol) and anti-Na/K ATPas (membrane) antibodies. The cytosolic fraction was shown in upper and the membrane fraction was shown in bottom. (d) p85 protein levels were determined from control or Myc-p42 (2(+) and 4(++) μg)-transfected HEK 293 cells by immunoblotting (upper), and quantification is shown as a bar graph (bottom). (e) Transfected PC12 cells were stained with an anti-p85 and Alexa Flour 594 goat anti-mouse antibody (gray) and visualized using a confocal microscope LSM 710 (Zeiss). The nucleus was counterstained with Hoechst (blue). Scale bar: 10 μm. (f) HEK 293T cells were transfected with different concentrations of Myc-p42 (2 or 4 μg); after 48 h, RT-PCR was performed with the extracted total RNA using p85-specific primers. (g) Cells transfected with control or GFP-p42 were incubated with cycloheximide (CHX; 10 μg/ml) for the indicated times. p85 protein levels were analyzed by immunoblotting (upper). Quantification of p85 protein levels in control (square) and GFP-p42 (circle) cells are shown (bottom); values are expressed relative to time 0 (normalized to actin). (h) HEK 293T cells transfected with SCR, N-si-p48 or si-Ebp1 were incubated with CHX (10 μg /ml) for the indicated times. p85 protein levels were analyzed by immunoblotting. (i) Control, myc-p42- or p48-transfected cells were used to determine protein levels of p85 and p110 by immunoblotting (left). Densitometry analysis (right). *P<0.05 versus control. (j) Endogenous p85 levels were determined from PC12 cells silenced with SCR, N-si-p48 or si-Ebp1 (left). Densitometry analysis (right). **P<0.005 versus control