Fig 1: PN/integrin β1-regulated Cdc42 activation is associated with stimulation of both the autophosphorylation and the kinase activity of Pak1 and actin cytoskeletal assembly in the VIC ex vivo cultures isolated from ED 17.5 mice mitral valves. (A) Upper panel: Cell lysates were harvested from growth-arrested VIC cultures that were either treated with or without 1 μg/ml of PN for 1 h (in serum-starved condition), or first treated with 5 μg/ml of both α5 and β1 integrin blocking antibodies for 2 h and then treated with 1 μg/ml of PN for 1 h. Cell lysates were prepared, and Cdc42-GTP was pulled-down from the lysates on GTP-loaded PBD-GST beads. The relative amounts of the activation of Cdc42 were quantified by normalizing the amount of bound Cdc42 to that of total Cdc42 in the lysates. Lower panel: Bars represent densitometric ratios of active Cdc42/total Cdc42 in the lysates. (B) Upper panel: Pak1 activation was tested by immunoprecipitation with anti-Pak and Western blotting with anti-p-Pak1 [Thr (T) 423]. The total amounts of p-Pak1 in the Pak1 IPs were determined by normalizing the amounts to total Pak1 by densitometry. Lower panel: Bars represent densitometric ratios of phospho p-Pak1 (T423)/total Pak1 in the lysates. (C) Upper panel: VICs were either treated with or without 5 μg/ml of both α5 and β1 integrin blocking antibodies for 2 h, or first treated with these blocking antibodies and then transfected with constitutively active (CA) Cdc42 cDNA for 48 h. PAK was immunoprecipitated from these treated/transfected VICs that were stimulated with 1 μg/ml PN for 5 min at 37°C, and a nonradioactive kinase assay was done using the traditional in-gel phosphorylation assay. For details of this experiment, see the “Materials and Methods” section. Pak1 kinase activities were evaluated by phosphorylation of substrate MBP (pMBP). The amounts of immunoprecipitated PAK were controlled by anti-PAK1 immunoblotting. Isotype control IgG was used as control. Lower panel: Bars represent densitometric ratios of active Cdc42/total Cdc42 in the lysates. (D) Upper panel: VICs were transfected with indicated lentivirus-expressing vectors. After 72 h, they were treated with and without PN for the indicated times. PAK was immunoprecipitated from platelets stimulated at indicated times at 37°C and incubated with 1 μg of MBP in the presence of 20 μM MgCl2-ATP and 0.185 MBq (5 μCi) γ[32P]-ATP (for details, see the “Materials and Methods” section). Autophosphorylated PAK was visualized as a 65-kDa phosphoprotein, and its kinase activity was evaluated by phosphorylation of MBP (pMBP). The amount of immunoprecipitated PAK was controlled by anti-PAK1 immunoblotting. Isotype control IgG was used as experiment control. Lower panel: PAK activation from the experiment of (D) was evaluated as in vitro kinase activity toward MBP, and quantification of PAK activity was done by densitometry analysis on ratio of expression of pMBP and total Pak1. (E) In vitro Pak1 kinase assays were further confirmed by nonradioactive assay kit (ab138879, Abcam), which is based on the monitoring of ADP formation (see “Materials and Methods” section for details). Data are presented as fold increase in Pak1 kinase activity with respect to control. (F,G) The effects of dominant negative mutants of N17 Cdc42 (DN Cdc42) and D57Y Cdc42 as well as the impact of blocking antibody for α5 and β1 integrin on actin dynamics were assayed. VICs were transfected with the indicated lentiviral constructs and incubated for 1 h with 10 μM G-actin, or in the presence or absence of 1 μg/ml of PN for 1 h. Lysates were assayed to determine actin dynamics by measurements of changes in the ratios (G) of F-actin/G-actin by immunoblot densitometry (F). The data in experiments in panels (A–G) are representative of ≥3 independent experiments. The values derived (A–G) from control cells were designated as 1.0. Error bars are reported as means ± SD of ≥3 separate experiments. *p < 0.05 was considered significant.