Mouse anti vinculin antibody clone hvin1

Bovine fibronectin was purchased from Sigma and used at 10 μg ml⁻¹ diluted in PBS (Sigma). Y-27632 dihydrochloride (Tocris Bioscience) was dissolved in water and used at 50 μM for 30 min. Mouse anti-vinculin antibody (clone hVin1) (Sigma, UK) was diluted (1:500) in 1% Bovine Serum Albumin (BSA) (cat: V9131, Sigma, UK). Dylight 594-conjugated AffiniPure Donkey Anti-Mouse IgG (cat: 715-585-150, Jackson ImmunoResearch, USA) was used as a secondary antibody, diluted in 1% BSA (1:500). Site-directed mutagenesis was performed using the QuikChange Lightning Multi Site-Directed Mutagenesis kit (Agilent, USA). For western blotting, the primary antibodies used were mouse anti-talin (8d4) (cat: T3287, Sigma) and mouse anti-GFP (cat: 11 814 460 001, Roche), diluted 1:500 and 1:250, respectively, in 2% milk (PBS 0.1% Tween). The secondary antibody was goat anti-mouse IgG conjugated to horseradish peroxidase (cat: A9917, Sigma), diluted 1:5,000 in 2% milk (PBS 0.1% Tween). Bands were detected using enhanced luminol-based chemiluminescent substrate (Promega).

Titanium sheet and diamond-coated silicon wafers
were cut into
2.2 × 2.2 cm² squares. The diamond films were terminated
with hydrogen and oxygen as described in Section 2.1.3. The substrates were sterilized in 70%
ethanol for 10 min and air-dried inside a laminar flow hood. The substrates
were adhered to the bottom of a reusable eight-well silicon insert
(flexiPERM^R, Heraeus Instruments). Cells were seeded at
the densities stated in Section 2.4.1 and cultured for 5 days. The medium
was changed every second day, and on day 5, cells were fixed with
4% paraformaldehyde for 15 min. Cells were then permeabilized with
0.2% Triton X-100, blocked with 4% BSA/4% FBS, and incubated overnight
at 8 °C with mouse antivinculin antibody (clone hVIN-1, Sigma-Aldrich).
The antivinculin antibody was detected with goat antimouse antibody-AlexaFluor
488 (Thermo Fisher Scientific). Cells were counter-stained with DAPI
and Phalloidin-Atto 565 (Sigma-Aldrich). Finally, substrates containing
the stained cells were mounted on #1.5 glass coverslips using Mowiol^R (Sigma-Aldrich) mounting media. Specimens were imaged using
a TCS SP8 confocal microscope (Leica Microsystems) equipped with hybrid
detectors, white and blue diode lasers, and a 40× immersion objective
(NA = 1.1). Whole volume images of the cells were acquired with a z-step of 0.5 μm.

The E64 cysteine protease inhibitor, serine protease phenylmethylsulfonyl fluoride (PMSF) inhibitor, and metalloprotease 1,10-phenanthroline (1,10-PT) inhibitor were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, 38070, France). Fluorescein phalloidin was obtained from Molecular Probes (Thermo Fisher Scientific, 91140 Les Ulis, France). Mouse anti-vinculin antibody (clone hVIN-1) was obtained from Sigma-Aldrich. Appropriate secondary antibodies were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, USA) and Molecular Probes Inc. (Thermo Fisher Scientific, Invitrogen Life Technologies, San Diego, CA, USA).