Fibronectin fragment (120 kDa cell attachment region) was from Merck (F1904).
IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10-fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. FRAX597 (S7271) and dasatinib (S1021) were obtained from SelleckChem, stock solutions were made in DMSO at 10 mM or 200 µM concentration, respectively, and diluted in culture media immediately before use. In all experiments, the cell density was adjusted to 3 × 105 cells/ml prior to treatment with inhibitors.
The antibodies used were the following: PAK1 (Cell Signaling, #2602), PAK1 (Abcam, ab223849), PAK2 (Abcam, #ab76293), PAK1 (phosphoS144)+PAK2 (phosphoS141)+PAK3 (phosphoS139) (Abcam, #ab40795), β-actin (Sigma-Aldrich, A5441), CD45-V450 (BD Biosciences, #560367), CD34-BV786 (BD Biosciences, #743534), Integrin β1-AlexaFluor647 (Abcam, ab193592), Integrin αVβ3-FITC (Abcam, ab93513). All PAK antibodies were previously characterized using siRNA [29 (link)].
IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10-fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. FRAX597 (S7271) and dasatinib (S1021) were obtained from SelleckChem, stock solutions were made in DMSO at 10 mM or 200 µM concentration, respectively, and diluted in culture media immediately before use. In all experiments, the cell density was adjusted to 3 × 105 cells/ml prior to treatment with inhibitors.
The antibodies used were the following: PAK1 (Cell Signaling, #2602), PAK1 (Abcam, ab223849), PAK2 (Abcam, #ab76293), PAK1 (phosphoS144)+PAK2 (phosphoS141)+PAK3 (phosphoS139) (Abcam, #ab40795), β-actin (Sigma-Aldrich, A5441), CD45-V450 (BD Biosciences, #560367), CD34-BV786 (BD Biosciences, #743534), Integrin β1-AlexaFluor647 (Abcam, ab193592), Integrin αVβ3-FITC (Abcam, ab93513). All PAK antibodies were previously characterized using siRNA [29 (link)].
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