Mouse monoclonal anti vinculin v9131

For FA staining, cells were seeded at a cell density of 15,000 cells/cm² and cultured for 24 h. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min, permeated with 0.2% Triton-100 in PBS for 5–10 min, and then blocked in 1% bovine serum albumin for 1 h. Samples were incubated with the primary antibody of mouse monoclonal anti-vinculin (v9131; Sigma-Aldrich, 1:400 dilution in 1% BSA) at 4°C overnight. After three times of wash with PBS, the samples were incubated with the secondary antibody of goat anti-mouse IgG-H&L FITC for 2 h (ab6785, Abcam, 1:1,000 dilution in PBS). Cell nuclei were stained using DAPI (500 ng/ml, Dojindo, D523, company, China).

Rabbit monoclonal anti‐N‐Myc (D1V2A) and rabbit polyclonal anti‐GAPDH (G9) were obtained from Cell Signaling Technology and Santa Cruz Biotechnology. Mouse monoclonal anti‐argonaute‐2 (Ago2) (EPR10411) was purchased from Abcam, and mouse monoclonal anti‐Vinculin (V9131) was procured from Sigma‐Aldrich. The cell lysate preparation and immunoblotting were performed using the methods described before (Challagundla et al., 2011, 2015).

To analyze the reorganization of Fn within the films, substrates were built using fluorescently labeled Fn (Fn*) with Alexa Fluor 568. A total of 5,000 HGF/cm² were seeded on the samples using complete medium and incubated at 37°C and 5% CO₂ during 2, 4, and 7 days, respectively, and immunostained as previously described (Section 2.7.5). For STED microscopy experiments, mouse monoclonal anti-vinculin V9131 (Sigma-Aldrich) diluted 1:400 in PBT (PBS + 0.1% Tween 20 + 2% BSA) was used to control focal adhesion formation, and the samples were incubated for 45 min at room temperature. Mouse Abberior STAR RED (Abberior, Göttingen, Germany) diluted 1:200 in PBT was added to the samples as secondary antibody and incubated for 1 h at room temperature. Substrates were washed with PBS (3×) for 15 min and mounted on microscope slides using Abberior Mount Liquid. Samples were examined using Abberior STEDYCON attached to a Zeiss Axio Imager.