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. 2015 Aug 22;6(30):29599–29613. doi: 10.18632/oncotarget.5011

Figure 3. RAD6, MDM2 and ASF1A form a ternary complex.

Figure 3

A. A two-step co-immunoprecipitation experiment was performed to test for ternary complex formation. HL-7702 cells were transfected with or without (no tag) Myc-RAD6 (RAD6A and RAD6B). The first immunoprecipitation was performed with an anti-Myc antibody. The complex was eluted using the Myc peptide. The second immunoprecipitation step used an anti-MDM2 antibody or the control mouse IgG (NIgG) to precipitate the complex. The protein samples from each step were then separately subjected to western blot analysis using anti-Myc, anti-MDM2, and anti-ASF1A antibodies. B. MDM2-induced ASF1A polyubiquitination is dependent on the presence of RAD6 in vivo. HL-7702 cells were transfected with (+) or without (−) Myc-MDM2 together with (+) or without (−) of RAD6 siRNAs for 48 h and incubated with 25 μM MG132 for another 10 h. Immunoprecipitation analysis was performed under denaturing conditions with an anti-ASF1A antibody. The anti-ASF1A antibody was also used to visualize the amounts of precipitated ASF1A and ubiquitinated ASF1A in the following western blot assay. The expression levels of ASF1A, Myc-MDM2 (Myc) and RAD6 are shown on the left.