TalinKO cells were transfected in a 6-well culture plate with either one of two siRNA sequences (Sigma) targeting mouse paxillin (paxillin siRNA 1, 5′-GUCGUAAAGAUUACUUCGA-3′; paxillin siRNA 2, 5′-CACUUUGUGUGCACCCACU-3′) using Lipofectamine 2000 as per the manufacturers’ instructions. After 48 h, one third of the cells were seeded onto fibronectin-coated glass in the presence of 5 mM Mn2+ and fixed 1 h after spreading. Cells were stained for paxillin (rabbit anti-paxillin, GTX125891; GeneTex) and imaged using the Zeiss AxioObserver Z1 wide-field microscope system, as described above, using a 40×/1.3 NA oil objective. Cell area was quantified manually using FIJI.
The remaining cells were lysed using RIPA buffer. 30 µg of protein was applied to SDS-PAGE, and proteins were transferred onto nitrocellulose membranes (Whatman). Membranes were blocked using casein blocking buffer (Sigma) and probed using primary antibodies diluted (1:1,000) in casein blocking buffer. Membranes were washed with Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 0.05% (vol/vol) Tween 20, followed by incubation with species-specific fluorescent dye–conjugated secondary antibodies (LI-COR Biosciences) diluted in PBS (1:5,000). Membranes were washed again and fluorescent signals were detected using the Odyssey infrared imaging system (LI-COR Biosciences).
The remaining cells were lysed using RIPA buffer. 30 µg of protein was applied to SDS-PAGE, and proteins were transferred onto nitrocellulose membranes (Whatman). Membranes were blocked using casein blocking buffer (Sigma) and probed using primary antibodies diluted (1:1,000) in casein blocking buffer. Membranes were washed with Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 0.05% (vol/vol) Tween 20, followed by incubation with species-specific fluorescent dye–conjugated secondary antibodies (LI-COR Biosciences) diluted in PBS (1:5,000). Membranes were washed again and fluorescent signals were detected using the Odyssey infrared imaging system (LI-COR Biosciences).