Fig. 3

p53 regulates Spy1 protein levels through the ubiquitin ligase Nedd4. a Western blot analysis of Spy1 (left panel) and p53 (middle panel) protein levels in MMTV-Spy1 primary mammary epithelial cells corrected for Actin. Data is represented as a fold change as compared to control siRNA (siCntl). Representative blot is shown in the right panel. b Levels of Spy1 protein were assessed via Western blot analysis 24 h after transfection in HC11 (n = 6) and MDA-MB 231 (n = 5) cells transfected with pEIZ, pEIZ-Spy1, p53 or both pEIZ-Spy1 and p53. Left panels depict representative blots and right panels depict densitometry analysis of Spy1 levels corrected for Actin. c Levels of Spy1 protein were assessed via Western blot analysis in the presence and absence of MG132. The left panel depicts representative blot, and the right panel depicts densitometry analysis of Spy1 protein levels corrected for Actin. Data is shown as fold change to cells transfected only with the Spy1 vector (n = 3). d Levels of Spy1 protein were assessed in HEK-293 cells after transfections with control vector pCS3 and Myc-Spy1-pCS3, p53, Skp2ΔF and Nedd4DN in various combinations. Cells were collected 24 h after transfection and subjected to Western blot analysis. Densitometry analysis was performed for total Spy1 protein levels and corrected for total Actin levels (n = 3). e Levels of Spy1 and Spy1-TST protein were assessed in HEK-293 cells after transfection with control vector pCS3, myc-Spy1-pCS3, myc-Spy1-TST-pCS3 and p53. Cells were collected 24 h after transfection and subjected to Western blot analysis. Densitometry analysis was performed for total Spy1 protein levels and corrected for total Actin levels (n = 3). Error bars represent SE; Student’s T test. *p < 0.05, **p < 0.01, ***p < 0.001, not significant (N.S.). See also Additional file 4: Figure S4