Fig 2: Nck (noncatalytic region of tyrosine kinase) 1 first SH3 domain regulates PAK2 (p21-activated kinase 2)-induced endothelial permeability. A, Schematic of Nck1 point mutations in each of the SH3 domains and the nomenclature used to inactivation of SH3 domains. B, Lysates from human aortic endothelial cells (HAECs) transiently transfected with Nck1 variants with inactivated SH3 domains. C and D, Flow-induced permeability using biotinylated gelatin/Alexa 647-streptavidin assay. 4',6-Diamidino-2-phenylindole (blue; nuclei), phalloidin (green; F-actin fibers), and streptavidin (red; exposed biotinylated gelatin) Images analyzed using NIS-Elements software from n=4. Scale bar=200 µm. Data were reported as box and whiskers with scatter plots and analyzed by Kruskal–Wallis test (*P<0.05,**P<0.01, ***P<0.001). E, Lysates and graphical quantifications from HAECs Nck1/2 double knockout (DKO) transiently transfected with either hNck1 or Nck1 SH3.1* and were immunoprecipitated for PECAM-1 (platelet endothelial cell adhesion molecule-1). Blotted for phospho-PAK2 S141. F, HAECs Nck1/2 DKO either transfected with hNck1 or Nck1 SH3.1* were lyzed after shear stress exposure (0–30 minutes) and immunoblotted for phospho-PAK2 S141 and GAPDH served as a loading control. Data are from n=3-4, expressed as mean±SEM, and analyzed by 2-way ANOVA and Bonferroni posttest (*P<0.05,**** P<0.0001).

Fig 3: Generation and analysis of conditional Rosa26-promoter-based expression alleles. a LR reaction performed between the pROSA26-DV1 vector and pEntry clone containing GST-PID* fragment to generate the Rosa26 targeting vector. SA is spice acceptor, PGK is phosphoglycerate kinase 1 promoter, and 3xpA is multimerized polyadenylation sequence in which loxP-PGK-neo-3xpA-loxP formed a LSL cassette. b Homologous recombination occurred between exon 1 and 2 of wild-type Rosa26 locus in G4 ES cells after electroporation. Black boxes represent the exons located at ROSA26 locus. c Knock-in targeted allele analyzed by PCR using both external primers (F1 and R1) and internal primers (F2 and R2). d Cre-mediated excision of intervening loxP flanked PGK-neo-3xpA (STOP) cassette resulted in the Rosa26-locus-based expression of an exon1-GST-PID*-IRES-eGFP bi-cistronic fusion transcript. e Representative result of genotyping PCR analysis of tail biopsy DNA detecting the presence of fusion transcript by both external primers (F1 and R1, 1.2 kb) and internal primers (F2 and R2, 1.4 kb), in which #54 and #70 were mice revealing positive results. f Phase contrast image (left side) and green fluorescence image (right side) showing the same field of view of MEF cells derived from PID mice after Adeno-cre virus infection. Representative western blot of GFP, GST, Pak1, Phospho-Pak expression level in MEF cells after infection of Adeno-cre virus. Relative protein expression was quantified and normalized to GAPDH