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. 2020 Nov 12;11(3):892–907.e1. doi: 10.1016/j.jcmgh.2020.11.001

Figure 4.

Figure 4

PAK1 associates and co-localizes with Notch1. (A) Immunofluorescence with staining for PAK1 (red) and Notch1 (green) in LBOs from WT mice showed co-localization in the cytoplasm. (B) Immunofluorescence analysis of SW480 cells transiently transfected with siPAK1 or siCTRL. Upper panel: siCTRL; middle panel: siCTRL + Jagged1 (100 ng/mL); and lower panel: siPAK1. In siCTRL SW480 cells, PAK1 and Notch1 co-localized in the cytoplasm. Treatment of SW480 cells with Jagged1 (Jag1) led to dissociation of PAK1 and Notch1 with increased nuclear Notch1. Silencing of PAK1 in SW480 resulted in similar observations, with nuclear translocation of Notch1. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Exemplary pictures of 2 independent experiments. (C) Western blot of SW480 cells after transient silencing of PAK1, showing up-regulation of activated Notch1. (D) Co-immunoprecipitation of SW480 and HCEC 1CT cells, using Notch1 or PAK1 as pull-down. Blotting for PAK1 and Notch1, respectively, indicated direct association of PAK1-Notch1, and β-catenin and Notch1. Associations were abrogated upon treatment with Jagged1. (E) Immunohistochemistry of NICD and PAK1 in AOM/DSS-treated WT and PAK1 KO mice. In PAK1 KO dysplastic lesions, NICD is highly up-regulated. PAK1 is up-regulated in inflamed-dysplastic lesions of WT mice. Results were analyzed for differences between the multiple independent groups by the Kruskal-Wallis ANOVA. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001, ∗∗∗∗P < .0001; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; IRS, immune-reactivity score; siPAK1, small interfering PAK1; siCTRL, small interfering Control; WCL, whole-cell lysate.