Phospho fak tyr397 pfak

The following primary monoclonal and polyclonal antibodies were used to detect cytoskeletal and FA proteins by immunofluorescent labeling and immunoblot analysis: anti-GAPDH monoclonal antibody (Cat. No. ab8245, Abcam, Cambridge, MA), anti-secretoglobin family 1A member 1 (CC10/scgb1a1; Cat. No. 213202, Abcam), anti-E-cadherin monoclonal antibody (Cat. No. 610181, BD Bioscience, San Jose, CA), total paxillin (PAX; Cat. No. 2542, Cell Signaling, Danvers, MA), phospho-PAX Tyr¹¹⁸ (pPAX; Cat. No. 2541, Cell Signaling), total focal adhesion kinase (FAK; Cat. No. 3285, Cell Signaling), phospho-FAK Tyr³⁹⁷ (pFAK, Cat. No. 3283, Cell Signaling), phospho-MLC2 (pMLC2; Cat. No. 3671t, Cell Signaling), MLC2 (Cat. No. 8505s, Cell Signaling), and RhoA (Cat. No. 2117s, Cell Signaling). Alexa Fluor 488 (Cat. No. A12379) and 633 (Cat. No. A22284) phalloidin as well as anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa Fluor 488, 568, or 633 (Cat. Nos. A-21206, A-10042, and A-21050) dyes were obtained from Thermo Fisher Scientific (Waltham, MA). The specificity of antibodies against MLC2 and pMLC2 has been reported previously (49 (link)–51 (link)). ROCK inhibitor Y-27632 was purchased from Millipore Sigma (Cat. No. SCM075, St. Louis, MO) and resuspended in deionized water. Y-27632 inhibits both ROCK1 and ROCK2 by competing with ATP for binding to the catalytic site (52 (link)).

Antibodies against the following proteins were used: RACK1 (1:1000, Cell Signaling, Danvers, MA, USA), phospho-Akt (Ser473) (p-Akt, 1:1000, Cell Signaling), Akt (1:1000, Cell Signaling), phospho-FAK (Tyr397) (p-FAK, 1:1000, Cell Signaling), FAK (1:1000, Cell Signaling), HA (1:1000, Cell Signaling), PHB2 (1:500, Santa Cruz, CA, USA), integrin β1 (1:500, Santa Cruz), β-actin (1:1000, Santa Cruz) and β-tubulin (1:500, Proteintech, Wuhan, China). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse/rabbit IgG) (1:5000, Cell Signaling) were used.

Cells were seeded at a density of 3 × 10⁴ cells/mL onto four different stiffness surfaces—1, 8, and 40 kPa hydrogels and a control 10 GPa plastic surface. After 24 h, the cells were fixed with 4% formaldehyde in PBS at RT for 10 min with gentle agitation (25 rpm). Samples were then rinsed twice with 0.05% Tween-20 (Merck KGaA) in PBS, permeabilised with 0.2% Triton X-100 in PBS at RT for 5 min, and cells were blocked with 3% BSA (Merck KGaA) and 10% FBS in PBS for 30 min. Next, cells were incubated with primary antibodies against phospho-FAK (Tyr397) (p-FAK) (Cell Signaling Technology, Inc., Danvers, MA, USA, Cat. No. 3283) protein. Antibodies were prepared in a blocking solution and cells were incubated for 1 h at RT. Then, samples were washed 3x for 5 min with 0.05% Tween-20 solution and incubated with secondary goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Inc., Cat. No. A-11008)—conjugated antibodies prepared in blocking solution in the dark for 1 h at RT. Samples were then washed 3x for 5 min with PBS at RT. In addition, cells were stained with 4 µg/mL (DAPI) prepared in PBS for 5 min in the dark at RT. Finally, samples were washed 3x for 5 min with PBS and visualised with a fluorescence microscope (Olympus IX51). The program ImageJ (1.8.0_112) was used for image processing.