Fig. 4
Identification of a nucleolar localization signal in SP110. a Amino acid sequence alignment of the wild-type and mutated nucleolar localization signals (NoLSs) of SP110. The sequence between amino acids 275 and 314 of SP110 identified as a potential NoLS is indicated in blue. Three lysine/arginine-rich (KR) clusters in the region are underlined. b and c HEK293T cells were transfected with the indicated constructs, and the localization of eGFP-SP1101–326 fusion proteins that contain the wild-type or mutated NoLS was visualized by confocal microscopy at 2 days post-transfection. The cells were probed with the anti-nucleolin antibody to identify nucleoli and subjected to Hoechst staining to identify nuclei. Scale bars: 10 μm. d HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative Luc values for the TNFα promoter were measured 2 days after co-transfection. The empty vector was included to the left as a negative control. The data are presented as the mean ± SD. Statistical significance of the difference between two sample groups was calculated using a two-tailed unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001. e HEK293T cells were transfected with the respective constructs, and whole-cell HEK293T extracts were analyzed by Western blot (WB) with the indicated antibodies at 2 days post-transfection. Red and black triangles indicate HA-SP1101–326 and β-ACTIN, respectively. f HEK293T cells were transfected with the indicated constructs, and the cellular distribution of eGFP-SP110b fusion proteins that contain a mutated NoLS was visualized using confocal microscopy at 2 days post-transfection (left panels). The cells were also subjected to Hoechst staining to identify nuclei (right panels). Scale bars: 10 μm. In (d and e), M1 + 2, M2 + 3 and M1 + 3 indicate Mut1 + 2, Mut2 + 3 and Mut1 + 3, respectively. All data represent at least 2 independent experiments