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. 2015 Mar 1;128(5):913–922. doi: 10.1242/jcs.158121

Fig. 1.

Fig. 1.

Identification of NLS sequences in Net1 isoforms. (A) Schematic of Net1 isoform domains and NLS sequences. Numbers refer to amino acids in mouse Net1 proteins. DH, Dbl homology domain; PH, pleckstrin homology domain; PDZ, PDZ-domain-binding site. (B) Subcellular fractionation of HeLa cells. Expression of endogenous Net1 and RhoA in the nuclear and cytosolic fractions was detected by western blotting. Lamin A/C and SOD1 were used as controls for purity of the nuclear and cytosolic fractions, respectively. Shown is a representative experiment from three independent experiments. (C) Subcellular localization of Net1 NLS mutants. HeLa cells were transfected with plasmids for HA-tagged wild-type and mutant Net1 cDNAs, fixed and stained for HA (green) and DNA (blue). Shown are representative images. (D) Quantification of subcellular localization of Net1 NLS mutants. Ratios are cytosolic staining divided by nuclear staining. Shown is the mean±s.e.m. of three independent experiments. At least 20 cells per experiment were analyzed. ***P<0.001. (E) Subcellular localization of Net1A NLS mutants in HeLa cells. Net1A proteins are in green, DNA is in blue. Shown are representative images. Scale bars: 20 µm. (F) Quantification of the subcellular localization of Net1A NLS mutants. Shown is the mean±s.e.m. of three independent experiments. ***P<0.001. cyto, cytoplasmic; nuc, nuclear.