Figure 7
From: Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression
Pin1 influences Notch3 processing and stability in endogenous and exogenous system. (a) Western blot analysis of Notch3 extracellular (N3EC) and activated intracellular (N3IC-act) protein expression of whole-cell extract (WCE) derived from Pin1-silenced TALL-1 (+) vs control cells (−) (left panel). The western blots in the figure are representative of at least three independent experiments, each in triplicate. The optical densitometry (OD) (right panels) was analyzed in all the experiments performed, thus including the P-values, calculated using Student’s T-test (i.e., **P⩽0.01). (b) WCEs from Pin1-silenced TALL-1 cells (+) vs control cells (−) in a time course assay with 10 μg/ml of cycloheximide (CHX), in the presence or absence of the proteasome inhibitor MG132 for the same times before lysis, were revealed by immunoblotting with anti-activated N3IC (N3IC-act), anti-Pin1 and anti-β-actin antibodies (left panel). The right panel shows the relative quantification of activated-N3IC as determined by OD. (c) Left panel, Western blot analysis of whole-cell extracts from HEK293T cells transfected with Flag N3IC-wt plasmid and silenced for Pin1 (+) or control (−) in a time course assay with 10 μg/ml of cycloheximide (CHX). Extracts were immunoblotted with anti-Flag, anti-Pin1 and anti-β-actin antibodies. The right panel shows the relative quantification of Flag N3IC as determined by OD. All data are representative of at least three independent experiments, each in triplicate.
