Skip to main content
. 2014 Oct 31;9(10):e111384. doi: 10.1371/journal.pone.0111384

Figure 2. ASPP2 regulates YAP phosphorylation level in vitro and in vivo.

Figure 2

(A) ASPP2 depletion leads to increased phosphorylated YAP at serine 127. ASPP2 was depleted in Caco-2 cells using siRNA and SDS-Page/immunoblotting was subsequently performed to analyse the expression level of the indicated proteins. An antibody specifically recognising YAP phosphorylated at serine 127 was used to analyse phosphorylation levels at this particular residue (A-E and G). (B) Par3 depletion does not affect the phosphorylation of YAP at S127. SiRNA knockdown was performed to deplete the indicated proteins and their expression was analysed by SDS-Page/immunoblotting. White arrowheads point to different Par-3 isoforms. (C) ASPP2 negatively regulates Lats-dependent phosphorylation of YAP at S127. SiRNA against ASPP2 and a combination of siRNA targeting Lats1 and lats2 were used as indicated in the figure. Four days following siRNA knockdown, protein levels were analysed by SDS-Page/immunoblotting. The ratios between pYAPS127 and YAP levels are indicated in the figure. (D-F) ASPP2 promotes the dephosphorylation of YAP at S127 in the colon. Exon 3 of ASPP2 was deleted by injection of tamoxifen in ASPP2 Δ3loxP-CreER mice. (D) Upper panel: YAP and pYAP S127 levels were analysed by SDS-Page/immunoblotting using lysates obtained from control and tamoxifen-injected mice (5 controls and 3 tamoxifen-injected animals). β-tubulin was used as loading control. Lower panel: RT-PCR showing deletion of exon3 of ASPP2 following tamoxifen treatment using RNA isolated from the colon of the same mouse. (E-F) Bar graphs representing the ratio between pYAP S127 and total YAP protein levels (E) and YAP protein expression levels normalised by β-tubulin (F). Data was normalised to the control. Error bars indicate standard deviation (*: p<0.05). (G) ASPP2 potentially induces the dephosphorylation of YAP at several serine residues. Following ASPP2 knockdown, lysates obtained from EJ cells stably expressing the indicated constructs were analysed by SDS-Page/immunoblotting using Phos-Tag (upper panel) or not (lower panel). Red arrowheads indicate differentially phosphorylated YAP proteins. The black arrowhead points to a non-specific band recognised by the anti-myc antibody (9E10).