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Figure 3


Fig. 3. Significance of the binding of afadin to nectin for the prevention of apoptosis. (A) Restoration of PDGF-induced phosphorylation of Akt by re-expression of full-length afadin, but not afadin{Delta}PDZ, in afadin-knockdown NIH3T3 cells. Four types of cells (control NIH3T3 cells, afadin-knockdown NIH3T3 cells, afadin-knockdown NIH3T3 cells re-expressing GFP-tagged full-length afadin and afadin-knockdown NIH3T3 cells re-expressing GFP-tagged afadin{Delta}PDZ) were treated with PDGF for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. The relative band intensity was calculated as described in Fig. 2A. *P<0.05 vs control NIH3T3 cells. C, control; KD, afadin knockdown. (B) Reduction of apoptosis by re-expression of full-length afadin in afadin-knockdown NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells stably expressing GFP, GFP-afadin or GFP-afadin{Delta}PDZ were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand for 24 hours with 3 ng/ml PDGF. Cells were immunostained with the anti-cleaved-caspase-3 mAb (red) and nuclei were counterstained with DAPI (blue). The graph represents the mean percentage of cleaved-caspase-3-positive cells in three independent experiments. In each experiment, the number of cleaved-caspase-3-positive cells among a total of 100 cells observed in four different fields of view was counted. Error bars indicate s.e.m. *P<0.05 vs control NIH3T3 cells expressing GFP. Scale bars: 50 µm. (C) Reduced expression of cleaved caspase-3 by re-expression of full-length afadin in afadin-knockdown NIH3T3 cells. Control NIH3T3 cells, afadin-knockdown NIH3T3 cells, afadin-knockdown NIH3T3 cells re-expressing GFP-tagged full-length afadin and afadin-knockdown NIH3T3 cells re-expressing GFP-tagged afadin{Delta}PDZ were cultured in the same conditions as described in B. Lysates from these cells were subjected to western blotting with the anti-cleaved-caspase-3 pAb. GAPDH was immunoblotted as a loading control.