Computational protocol: Kindlin-2 directly binds actin and regulates integrin outside-in signaling

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Protocol publication

[…] Cell spreading assays were performed with MAE, C2C12, and OVCAR-3 cells. The cells were either untransfected or transiently transfected for 24 h and allowed to adhere and spread on vitronectin-coated (2.5 µg/ml) or fibronectin-coated (20 µg/ml) glass coverslips. After incubation at 37°C for selected times, the coverslips were washed three times with PBS, and the adherent cells were fixed with 4% paraformaldehyde and stained with Alexa Fluor–phalloidin (Life Technologies). As controls, untransfected cells were included in each experiment. For ILK and K2 localization in C2C12 cells, cells were costained with anti-vinculin monoclonal antibody, followed by Alexa Fluor 568–conjugated F(ab′)2 anti-mouse IgG secondary antibody. The coverslips were mounted with ProLong Gold antifade reagent (Life Technologies). The positively stained cells were visualized at 40× or 63× using a TCS-NT laser scanning confocal microscope (Leica), using confocal software (Leica). Images shown are representative of at least three independent experiments. Cell areas were analyzed with ImageJ software. [...] Means and standard errors are reported. Statistical analyses using a two-tailed Student’s t tests were performed in SigmaPlot (version 11). Differences were considered to be significant with P < 0.05. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Neoplasms by Site