Figure 6
From: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression
Pin1 affects Notch3 processing. (a) CD4+ and/or CD8+ subset distribution of thymocytes from Pin1+/+ and Pin1−/− mice. In both panels, numbers inside each cytogram indicate the percentages of the corresponding subsets. (b) RT–PCR shows the unchanged relative Notch3 mRNA levels in Pin1−/− vs Pin1+/+ thymocytes (left panel). (Right panel) Western blot analysis of whole-cell extracts (WCEs) from the same thymocytes probed with anti-Notch3EC (N3EC) and anti-Pin1 antibodies. The β-actin expression was used as a loading control. (c) Notch3 extracellular expression (N3EC) from thymocytes of Pin1+/+ and Pin1−/− mice indicated as percentages inside each cytogram. The violet curve represents the isotypic control. The mean fluorescence intensity (MFI) ratio between Notch3 and isotypic control staining is also indicated. The results showed in both panels are representative of five independent experiments (n=5 mice for group). (d) Bar graphs represent the absolute cell number from thymocytes expressing N3EC of the same mice indicated in (c). (e) Cytosolic (C) and membrane (M) fractions from Pin1+/+ and Pin1−/− thymocytes were analyzed in immunoblot assays to detect the N3EC expression. Anti-Lck and anti-α-tubulin were used as fraction markers; anti-β-actin was used as a loading control. (f) Thymocytes from Pin1+/+ and Pin1−/− mice were incubated with EZ-Link Sulfo-NHS-SS-Biotin (+) or were mock (−) treated, as described in Materials and methods. Cells were lysed and extracts were loaded on a 6% SDS–PAGE gel either directly (T fraction, 15% of the extract) or after incubation on streptavidin-agarose beads (B fraction, 85% of the extract). Extracts were then immunoblotted with the anti-N3EC and anti-N3IC antibodies. Positions of the 210-kDa Notch3 extracellular (EC) and 97-kDa Notch3 transmembrane-intracellular (TM-IC) domains are indicated by black arrows. In the high exposition is indicated the position of the Notch3 intracellular domain (IC) (red arrow). ^ indicates non-specific bands. (g) Nuclear fractions from Pin1+/+ and Pin1−/− thymocytes were analyzed in immunoblot assays to detect the N3IC expression. Anti-LaminB and anti-α-tubulin were used as fraction markers; anti-β-actin was used as a loading control. In all panels (b) and (d), results are shown as the means average deviations of five separate experiments and P-values were calculated using Student’s T-test (i.e., ns, not significant P>0.05; **P⩽0.01). In all the western blots represented in the figure, FL indicates Notch3 full-length receptor and EC indicates extracellular region.
