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. 2011 Nov 2;6(11):e26954. doi: 10.1371/journal.pone.0026954

Figure 5. Subcellular localization of Nocturnin protein.

Figure 5

(A) Nocturnin protein does not co-localize with stress granules (SGs) or P-bodies (PBs). NIH3T3 cells were transfected with NOC-FLAG only (arsenite-induced SG and PB experiments) or pE-eGFP-G3BP1 and NOC-FLAG (G3BP1-induced SG experiments) constructs. Specific antibodies were used to detect NOC-FLAG and GE-heds (marker for P-bodies). Part of the image was amplified on the right to more clearly show the lack of overlap. Shown are representative examples from 3 independent experiments. (B) Nocturnin protein does not co-localize with polysomes. Polysome analysis was conducted by sucrose gradients. EDTA or cycloheximide was utilized to disassociate or freeze the polysomes, respectively. Fractions were collected and separated by SDS-PAGE and analyzed by Western blotting. Tubulin and ribosomal P antigen were blotted as markers of non-polysome fractions and ribosome/polysome fractions, respectively. This fractionation was performed on two independent occasions with indistinguishable results.