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Figure 3


Fig. 3. Generation of normal traction forces by cells defective in calpain 1 or calpain 2. (A) Average integrated traction forces generated by MEF cells treated with siRNA for either Capn4, Capn1 or Capn2 or cells overexpressing calpastatin. Neither silencing of calpain 1 or calpain 2, nor overexpression of calpastatin, causes a detectable reduction in traction forces. Data are compiled from measurements of a minimum of 15 cells for each cell type. Although wild-type MEF cells transfected with siRNA against Capn4 show a significant reduction in the magnitude of traction stress (Student's t-test P=0.0001) (n=19). (B,C) Immunofluorescence of the calpain small subunit in MEFs transfected with control RNA (B), or with siRNA against Capn4 (C) shows a striking reduction in the amount of small subunit upon siRNA-mediated gene silencing. (D) RT-PCR demonstrates an 88% reduction in the amount of Capn4 mRNAs. (E) Level of calpastatin protein expressed in wild-type MEF cells transfected with GFP plasmid alone and cells in which calpastatin-GFP has been overexpressed.





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