Different Modes of Retrovirus Restriction by Human APOBEC3A and APOBEC3G In Vivo
Figure 2
Cellular lysates from splenocytes derived from uninfected transgenic, wild type (BL/6) or mA3 knockout (KO) mice or from 293T cell lines over-expressing A3A or A3G were incubated with a 3′-fluorophore labeled 50-mer single-stranded oligonucleotide (S50) containing cytosine in the sequence context preferred by A3G (S50-CCC) or A3A (S50-TTC).
Deamination was detected by uracil excision by UDG followed by fragmentation of the resulting abasic site by NaOH and heat, resulting in a 35-mer product (P35). High levels of activity in the 293/A3A samples result in deamination at multiple potential cytosines in the S50-CCC substrate (P37). This experiment was performed several times with the same lysates, with similar results.