Analysis of insulin-regulated phosphorylation of AFF4 with Akti or S6ki and S6K invitro kinase

Aff4 was recently identified as an insulin-regulated phosphoprotein. To investigate upstream kinases, we analysed immunoprecipitated Aff4 treated with our without insulin and treated with either an Akti or a P70S6Ki. These results showed that AFF4 is dependent on P70S6K and therefore an in vitro kinase assay was performed to validate direct phosphorylation.