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Fig. 5. Exogenous FRNK expression induces keratinocyte apoptosis on LN-5 ECM. (A,B) Wild-type MK+/+ cells were left uninfected (uninfect. lanes) or infected with adenovirus expressing either GFP-FRNK fusion protein (GFP-FRNK lanes) or GFP only as a control (GFP lanes). Cells were then sub-cultured on LN-5 ECM for 24 hours under serum-free conditions. (A) Cell lysates were immunoblotted with anti-GFP antibody to confirm expression of exogenous proteins; the migratory positions of GFP (27-29 kDa) and GFP-FRNK (
68 kDa) are indicated. (B) Cells were assayed for activation of caspase-3 by immunoblotting as described in Fig. 2; migratory positions of pro-caspase-3 and cleaved caspase-3 are indicated. A lower proportion of intact protein was recovered from GFP-FRNK expressing cells due to extensive apoptosis (see panels Cc,d); therefore, a longer exposure of the GFP-FRNK lane is included to emphasize the proportion of caspase-3 that is cleaved. Filters were stripped and reprobed for keratin 14 (K14). (C) Fluorescence of representative fields of cells infected with either GFP (a) or GFP-FRNK (c). Corresponding phase images are shown in b and d, respectively. Arrowheads point to examples of infected (i.e. fluorescent) cells. Note that most cells expressing GFP remained well spread with no signs of apoptosis (a,b), while the vast majority of GFP-FRNK expressing cells displayed an apoptotic phenotype (c,d). Arrows in panels c and d point to uninfected cells, which do not show signs of apoptosis and serve as an internal control.
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