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. 2012 Sep 28;31(21):4236–4246. doi: 10.1038/emboj.2012.271

Figure 2.

Figure 2

Expression of ZAP excludes target mRNA out of polysome. (A) 293TREx-ZAP cells were infected with VSV-G pseudotyped NL4-3-luc virus for 3 h, and mock-treated (Tet−) or treated with 1 μg/ml tetracycline (Tet+) for additional 48 h to induce ZAP expression. Cells were then treated with 50 μg/ml cycloheximide to stop global translation. Cell lysates were resolved in a 10–50% continuous sucrose gradient by ultracentrifugation and polysome profiles were obtained by monitoring absorbance at 254 nm. Nef-luc and GAPDH mRNA levels in each fraction were determined by RT–PCR, followed by Southern blotting. (B) 293TREx-ZAP/FL-Mk cells were mock-treated (Tet−) or treated with 1 μg/ml tetracycline (Tet+) for 48 h to induce ZAP expression, followed by treatment with 50 μg/ml cycloheximide to stop global translation. Cell lysates were resolved in a 10–50% continuous sucrose gradient by ultracentrifugation and polysome profiles were obtained by monitoring the absorbance at 254 nm. Luc-Mk and GAPDH mRNA levels in each fraction were determined by RT–PCR. Figure source data can be found with the Supplementary data.