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Figure 3 | Retrovirology

Figure 3

From: APOBEC3G-UBA2 fusion as a potential strategy for stable expression of APOBEC3G and inhibition of HIV-1 replication

Figure 3

Fusion of UBA2 to APOBEC3G limits its polyubiquitination. A-a, Expression of polyubiquitin abolishes the ability of UBA2 to stabilize APOBEC3G. HEK293 cells were co-transfected as described in Fig. 2. In addition, 3 μg of a plasmid DNA (pcDNA3.1-HA-Ubiquitin) that produces polyubiquitin [40, 41] was also co-transfected to HEK293 cells. Western blot analysis was carried out by using monoclonal anti-APOBEC3G, anti-Vif, anti-HA, and anti-β-actin antibodies respectively. A-b. The intensity of APOBEC3G protein and value of the relative intensity of APOBEC3G was determined as described in Fig. 2. Note, a protein band that migrates with similar size to APOBEC3G-UBA2 as shown in lane E sometimes react to anti- APOBEC3G antibody. This is a non-specific protein band because it only reacts to certain batches of anti-APOBEC3G antibody. To eliminate this background, the protein intensity of APOBEC3G-UBA2 and APOBEC3G-UBA2* was calculated by subtracting the level of this non-specific protein. B. Fusion of UBA2 to APOBEC3G shows reduced polyubiquitination. B-a, Vif protein was pull-down in different APOBEC3G-producing HEK293 cells by immunoprecipitation using anti-Vif antibody. Binding of high molecular weight of ubiquitin (polyubiquitin) to Vif was detected by using anti-ubiquitin antibody. B-b, the relative intensity of ubiquitin to β-actin control was determined by densitometry. Also note that there are not much protein level differences of APOBEC3G between lane 2 (U) and lane 3 (M). This is likely due to the fact that more protein is loaded in lane 3 than lane 2 as shown by the relative protein levels of β-actin.

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