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. 2011 Jul 15;124(14):2478–2487. doi: 10.1242/jcs.078170

Fig. 5.

Fig. 5.

4.1R is required for actin stress fiber and focal adhesion formation in keratinocytes. (A) Endogenous actin filament staining. Keratinocytes were cultured on fibronectin-coated surface for 36 hours, the cells were then fixed and stained for actin using Rhodamine–phalloidin. (B) Exogenously transfected GFP–actin. Keratinocytes were transiently transfected with GFP–actin. 24 hours after transformation, samples were fixed and viewed. All images shown were collected by wide-field epifluorescence microscopy, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Immunostaining of focal adhesion proteins. Cells were stained for β1 integrin, talin, paxillin and vinculin. All samples were counterstained for actin with Rhodamine–phalloidin (red) and DAPI (blue). Scale bars: 20 μm. (D) Localization of transiently transfected GFP–vinculin in keratinocytes. EGFP–vinculin was transiently transfected into 4.1R+/+ or 4.1R−/− primary keratinocytes using Fugene6. GFP-positive cells were sorted by FACS. Cells were viewed for fluorescence using a Zeiss Axiophot wide-field epifluorescence microscope. (E) Western blot analysis of actin and focal adhesion proteins. Total lysates (20 μg protein) were probed with indicated antibodies. GAPDH immunoblot is shown as a loading control. (F) Quantitative analysis from three independent experiments.