α actin

The WG-59 cell characterization was performed by flow cytometry at the 20th passage. In brief, 100 µL of 1 × 10⁶ cells were incubated for 20 min at 4 °C in BD Cytofix/Cytoperm (BD Bioscience, Heidelberg, Germany) in order to fix and the cells and permeabilize the cell membrane. Cells were then washed with 10% BD Wash/Perm Solution (BD Bioscience) and incubated with antibodies against α-actin (R&D Systems, Minneapolis, MN, USA), myosin heavy chain (R&D Systems, Minneapolis, MN, USA), calponin (Acris, San Diego, CA, USA) and smoothelin (Acris, San Diego, CA, USA) in a total volume of 50 µL for 30 min. Cells were washed again in 10% BD Wash/Perm Solution and were resuspended in 300–400 µL BD Staining Buffer (BD Bioscience). The measurement was performed on a benchtop analyzer LSR II (Becton Dickenson, Heidelberg, Germany). FlowJo software (Tree Star, Inc., Ashland, OR, USA) was used for data analysis. To exclude non-specific binding, mouse IgG1 and IgG2a isotype controls (BioLegend, London, UK) were used.

ECFCs (6-well plate, 500,000 cells/well) were lysed in SDS denaturing buffer (50 mM Tris, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM β-glycerophosphate, 100 μM phenylarsine oxide, 1% SDS, 5 μg/mL leupeptin, 10 μg/mL aprotinin, pH 7.4). Proteins were subjected to SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with various primary antibodies to phospho-cofilin (Ser 3) (Cell Signaling, MA, USA #3311, rabbit antibody, 1/1000), cofilin (Cell Signaling, MA, USA #5175, monoclonal rabbit antibody, 1/1000), α-actin (R&D, UK #MAB8929, mouse monoclonal antibody, 1/40,000), α-tubulin (Abcam , Cambridge, UK #11304, mouse monoclonal antibody, 1/2000), acetylated α-tubulin (Sigma #T7451, mouse monoclonal antibody, 1/10,000), tyrosinated α-tubulin (Millipore #ABT171, rabbit, 1/2000), detyrosinated α-tubulin (Abcam #48389, rabbit, 1/1000) and Eng (mouse monoclonal antibody; P4A4) [11 (link)]. Secondary antibodies used were HRP-conjugated goat anti-mouse (Jackson Immunoresearch, PA, USA #115-035-003, 1/20,000) and HRP-conjugated goat anti-rabbit (Jackson Immunoresearch PA, USA #111-035-144, 1/20,000). Immunoreactive bands were visualized using Enhanced Chemiluminescence Detection Reagents (Pierce). Images of the chemiluminescent signal were captured using G:BOX Chemi XT16 Image Systems and quantified using ImageJ 1.53 software.