Figure S3.

Additional data to Fig. 3. (A) The two breast cancer cell lines, MCF-7 and Cal51 used in Fig. 3, H and I, were grown as xenografts and stained for Numb and pNumb in IHC, in comparison to two PDXs from Numb-competent tumors displaying low pNumb (BC #4) and high pNumb (BC #2; the BCs in object are described in details in Fig. 8) to provide additional proof that they are representative of the situation occurring in real breast cancers. Bar, 100 μm. (B) The phosphorylation of Numb executed by PKCζ does not appreciably affect the half-life of Numb. HEK-293 cells were transduced with Venus (EV) or PKCζ-Venus (PKCζ -OE), as shown on the top, and then treated with cycloheximide (CHX, 50 μg/ml) for the indicated lengths of time. Lysates were immunoblotted as shown (top four panels). Mdm2 was used as control for a short half-lived protein. GAPDH is a loading control. In the lower panel (Phos-tag), the lysates were fractionated on an 8% Phos-tag gel (see Materials and methods) and then IB with anti-Numb. The nearly complete shift on Numb in the PKCζ-OE cells indicates that the phosphorylation of Numb executed by PKCζ is nearly stoichiometric. Source data are available for this figure: SourceData FS3.