Expression and cell membrane localization of heterologous β1 integrin.
(A) Quantitative RT-PCR for quantification of β1 integrin mRNA performed on total mRNA extracted from cultured keratinocytes derived from a non-transgenic pig (#2990) and the six hITGB1 transgenic pigs, normalized to endogenous β-actin. The expression level of human β1 integrin was proportional to genomic integration copies and only detectable in the transgenic animals. Data are presented as mean values ± standard deviations. (B) Western blot analysis on total cellular protein from the keratinocytes described in (A). Employing the mouse anti-human β1 integrin mAb (610467), both pre- and post-translationally processed human β1 integrin protein is detected in all transgenic pigs. ACTB detection was used as loading control. (C) Flow cytometry analysis of keratinocytes stained with an anti-human β1 integrin mAb (P5D2) without permeabilization (light gray bars) or with saponin permeabilization (dark gray bars). Representative histograms comparing fluorescence from pig #2990 and #3404 with and without permeabilization are depicted. In five out of the six hITGB1 transgenic keratinocytes, β1 integrin was predominately localized to the cell membrane. (D) Representative images from wide-field (a–b) and confocal (c–d) fluorescence microscopy of permeabilized keratinocytes (derived from the non-transgenic control (#2990) and hITGB1 transgenic pigs) stained with anti-human β1 integrin (P5D2). Detection of β1 integrin was restricted to transgenic cells with accumulation at the plasma membrane and especially at cell-cell adhesion sites.