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Figure 2 | Retrovirology

Figure 2

From: Characterization of APOBEC3G binding to 7SL RNA

Figure 2

APOBEC3G binds 7SL RNA. A. Predicted secondary structure of Alu and 7SL RNAs. The latter comprises Alu and S domains. Bases in red are identical between the Alu used in this study (belonging to Alu Sb1 family) and 7SL. For competition experiments (see 2D), the indicated Alu and S stem domains of 7SL RNA were used. B. Indicated HA-tagged cytidine deaminases were immunoprecipitated. The resulting material was analyzed by HA-specific western blot (top) and 7SL RNA-specific standard (middle) or real-time (bottom) RT-PCR. For APOBEC3G, indicated three doses of plasmid were transfected; for all the others, 20 μg of DNA were used. NT, non-transfected. C. Alu RNA competes with 7SL RNA for binding to APOBEC3G. Increasing doses of Alu-Sb1 RNA-expressing plasmid were co-transfected with a fixed amount of APOBEC3G DNA. Upper panel: APOBEC3G in total cellular homogenates in one representative experiment. PCNA was blotted as a loading control. Bottom panel: APOBEC3G was immunoprecipitated and Real Time PCR was used to quantify levels of 7SL RNA in the immunoprecipitates. Means and SE from three independent experiments are shown. D. The Alu domain of 7SL RNA competes with full-length 7SL RNA for APOBEC3G binding. Alu-Sb1 RNA or 7SL Alu and S stem domains-expressing plasmids were co-transfected with APOBEC3G DNA at a 3:1 ratio. Upper panel: APOBEC3G in total cellular homogenates in one representative experiment, PCNA serving as a loading control. Bottom panel: 7SL RNA-specific real time PCR on APOBEC3G-specific immunoprecipitates. Means and SD from two independent experiments are shown.

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