FIGURE 6:  Phosphorylation of the FUS prionlike domain is not linked to cytoplasmic localization. (A) H4 cells following calicheamicin treatment, with and without DNA-PK inhibitor (NU7441), were fixed for immunocytochemical analysis using commercial FUS and anti-FUS(pSer30) antibodies; n = 3. (B) Quantification of nuclear and cytoplasmic fluorescence signals was done to determine the percentage of cytoplasmic localization; error bars represent 95% confidence interval. (C) Lysates were analyzed by Western blotting; n = 3. (D) H4 cells expressing ectopic PM FUS (6 or 12 glutamate substitutions in the prionlike domain) were visualized by immunofluorescence microscopy; n = 3. (E) Quantification of nuclear and cytoplasmic fluorescence signal was done to determine the percentage of cytoplasmic localization of FUS-wt and FUS-12E from C; error bars represent 95% confidence intervals. (F) U-2 OS cells expressing GFP-fused ectopic phosphomimetic FUS were visualized by live-cell imaging; n = 2.