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

Figure 2. MDM2 regulates ASF1A ubiquitination and degradation in Homo sapiens.

Figure 2

A. MDM2 interacts with ASF1A and RAD6 in vivo. HL-7702 cells were lysed for an endogenous Co-IP assay with an anti-MDM2 antibody. Western blot assays were performed using anti-ASF1A or anti-RAD6 antibodies to detect the interaction between MDM2 and ASF1A or MDM2 and RAD6 in vivo. B. MDM2 colocalizes with ASF1A in HL-7702 cells. HL-7702 cells transfected with MDM2-Myc and ASF1A-Red were used for an immunofluorescence assay with an anti-myc antibody (green). C. MDM2 promotes ASF1A degradation. HL-7702 cells were transfected with or without HA-tagged MDM2 for 48 h. The cells were then incubated with 50 μg/mL CHX for the indicated times. Cell lysates were prepared and subjected to western blot assays with antibodies as indicated. D. MDM2 regulates ASF1A ubiquitination. HL-7702 cells were transfected with control or MDM2-specific siRNA for 48 h as indicated. The cells were then incubated with 25 μM MG132 for another 10 h. An immunoprecipitation experiment was performed using an anti-ASF1A antibody under denaturing conditions. An anti-ASF1A antibody was used in a western blot assay to detect the ubiquitinated form of ASF1A. E. MDM2 regulates ASF1A protein levels and H3K56Ac levels through the 26s proteasome. HL-7702 cells were transfected with an HA-tagged MDM2 plasmid or a control HA empty vector or with MDM2-specific siRNA or control siRNA for 48 h. The cells were then lysed for western blot assays with antibodies as indicated (upper). HL-7702 cells were transfected with HA-tagged MDM2 or MDM2-specific siRNA for 48 h. Total RNA was isolated and subjected to an RT-PCR assay with primers specific for asf1a and gapdh genes (lower).