Fig 1: Nucleolar localization of eIF6 is not dependent on its interaction with BCCIP or uL14. (A) Western blot represents the nuclear (nuc) and cytoplasmic (cyto) distribution of eIF6. Blots were probed with anti-eIF6 (Santa Cruz Biotechnology), anti-topoisomerase II-β (nuclear marker) and anti-β-tubulin (cytoplasmic marker) antibodies. Adjacent western blots represent the total input levels of eIF6 and β-tubulin (loading control). (B and C) Blots shown in panel (A) were quantitated and the percent distribution of eIF6 in nuclear and cytoplasmic fractions were plotted for eIF6-WT (B) and eIF6-N106S mutant (C). Plots indicate the standard error of the mean of three independent experiments. (D) IF images of asynchronous cells show localization of eIF6 to nucleoli (arrows) in both WT and mutant cells and decreased nuclear localization in mutant. Fixed cells were stained with DAPI (nuclear marker) or with anti-eIF6 antibody. Scale bar highlighted in the top left image represents scale bars for all images shown in panel. (E) Plot indicates standard error of the mean of eIF6 fluorescence intensity in nucleus normalized to DAPI intensity per cell. Significant difference (P= 0.03) determined using an unpaired two-tailed t-test. (F) Representative western blot shows IP of endogenous eIF6 or anti-mouse IgG control and its interaction with endogenous BCCIP. The corresponding input is shown. Vinculin used as loading control. (G) Representative western blot shows IP of endogenous BCCIPβ or anti-rabbit IgG control and its interaction with endogenous eIF6. The corresponding input is shown. Vinculin used as loading control. (*Heavy-chain IgG migrates close to BCCIPβ).