Fig. 7

The effects of mutating either the NLS or the adjacent S1006 upon the degree of nuclear localization of hPPIP5K2. Panel a shows Western analysis of extracts of HEK293T cells in which various hPPIP5K2-GFP constructs were expressed. For clarity, only four of the molecular markers are labeled. Each of the lanes transfected with a hPPIP5K-GFP construct showed a faint immunoreactive band that corresponded to the migration of GFP alone. ImageJ analysis indicated this represented <2% of the total signal, which is too small to compromise any of our conclusions. Panel b shows representative images of live HEK293T cells that expressed each of the various constructs. The size bar represents 10 μm. From each group, fifteen cells were randomly selected and F_N / F_C ratios were calculated (panel c); data represent means ± SEM (** p < 0.001, *p < 0.02 compared to wild-type (WT), determined using Student's t-test). Imaging flow cytometry was also used to analyze fixed and DRAQ5 stained HEK293T cells that were transfected with either d, hPPIP5K2-GFP (n = 2357; 57719 ± 1116 total fluorescence units); e, hPPIP5K2^NLS-3A-GFP (n = 2179; 52386 ± 907 total fluorescence units); f, hPPIP5K2^S1006A-GFP (n = 1686; 50383 ± 947 total fluorescence units); g, hPPIP5K2^S1006D-GFP (n = 1696; 57991 ± 1162 total fluorescence units). The histograms depict the frequency of binned. Similarity Scores denoting the degree of co-localization of the GFP signal and nuclear stain, derived on a cell-by-cell basis. Best-fit Gaussian plots are also shown. The blue Gaussian plot for hPPIP5K2-GFP is superimposed upon the other plots for comparative purposes. Statistical differences in median similarity scores (“M”) compared to hPPIP5K2-GFP were determined using a Mann–Whitney Rank Sum Test. Note that the Western analysis includes an extract from cells transfected with a hPPIP5K2^S1006E-GFP construct. The Similarity Score of the latter (not shown) was not significantly different from that of hPPIP5K2^S1006D-GFP