Mutational analysis of putative NLSs in H-TWIST were observed in U2-OS cells, the expression of c-myc-TWIST mutants were detected by fluorescence using anti c-myc antibody. Nuclei were stained with DAPI (blue), and phalloidin (green) representing the actin cytoskeletal. Site-directed mutagenesis generated a single amino acid change, K38R, which was sufficient to block TWIST nuclear localization and, therefore, redistribute approximately 92% the mutant protein to the cytoplasm. Mutations in TWISTNLS2 at positions K73R and K77R also resulted in cytoplasmic expression, as approximately 83% and 87% of transfected cells had very low levels in the nucleus. (b) Analysis of expression levels of TWIST mutant constructs in cytoplasmic and nuclear fractions of transfected cells. Equivalent loading was determined by GAPDH and α-actin expression levels.