Ab32084

Cells were fixed with 3.6% (v/v) paraformaldehyde (Sigma-Aldrich) in PBS for 20 min at room temperature. To block and permeabilize, fixed cells were incubated in 2% (w/v) BSA + 0.1% (v/v) triton X (VWR) in PBS. Zyxin or paxillin (Abcam, ab58210 and ab32084, respectively, both 1/1000) were incubated in 2% (w/v) BSA + 0.05% (v/v) tween-20 in PBS overnight at 4 °C. The following day, 1/1000 Goat-anti-mouse Alexa Fluor 568 or Goat-anti-rabbit Alexa Fluor 488 was incubated overnight at 4 °C in 2% (w/v) BSA + 0.05% (v/v) tween-20 in PBS. The next day, samples were stained with DAPI (Sigma-Aldrich, 0.14 µg/ml in PBS + 0.05% (v/v) tween-20) to stain nuclei. Images were taken on a confocal microscope.
Focal adhesions were quantified manually by counting the number of focal adhesions per cell using Fiji. Between 17 and 27 cells were counted per condition, from 5 to 10 different images from biological triplicates. Cell area was measured by manually outlining the cells and measuring surface area using Fiji. The number of focal adhesions was normalized to the cell area.

Cells (5 × 10³) were seeded in a 12-well plate paved with sterile slips. After 24 h, the cells were fixed, permeabilized, blocked, and then incubated with anti-paxillin antibody (ab32084, Abcam, USA) at a dilution of 1:150 overnight at 4 °C. The next day, the slips were incubated with fluorescently labeled secondary antibody and phalloidin (ab176756, Abcam, USA) for 2 h at room temperature. Subsequently, DAPI was used to stain nuclei. Finally, fluorescence was observed and imaged under the confocal laser-scanning microscope (LSM880, Zeiss, Germany).

Whole cell lysates were run on 4-16% SDS-PAGE gels (ThermoFisher) and transferred to PVDF membranes (Invitrogen). Antibodies used were: anti-HNF1B (H-85) (SC-22840, Santa Cruz), anti-B-actin (AC-15) (ab6276, Abcam), anti-paxillin [Y-113] (ab32084, Abcam), anti-integrin α2 (611016, BB Biosciences), anti-integrin β1 (610467, BD Biosciences).

Fixed cells were incubated for 10 min with 0.25% Triton X-100 followed by 1% albumin overnight at 4°C for blocking. Primary paxillin antibody (1∶2000, ab32084, Abcam) was applied for 2 hours at room temperature, and then a secondary AlexaFluor 488-conjugated antibody (1∶2000, Invitrogen) was applied for 1 hour or rhodamine phalloidin (1∶2000 Invitrogen) and Hoechst 33342 (3.2 µM, Invitrogen) for 30 min at room temperature. The cells were subsequently mounted with Fluoromount-G (Southern Biotech, Birmingham, AL). All buffers used contained 1 mM MgCl₂. The samples were imaged by using a CARV II confocal (BD Biosciences) Nikon Eclipse Ti-S microscope equipped with a motorized, programmable stage using a Cool-Snap HQ camera (Photometrics) and controlled by Metamorph 7.6 (Molecular Devices). A custom-written MATLAB (Mathworks) program was used to quantify cell area and focal adhesion number and size.