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. 2013 Jun 7;8(6):e65330. doi: 10.1371/journal.pone.0065330

Figure 2. E6AP downregulates expression of proadipogenic factor C/EBPα.

Figure 2

(a,b) 3T3-L1 preadipocytes were transfected with increasing amounts of E6AP (0.5 µg–2.0 µg) and E6AP-C843A (0.5 µg–2.0 µg). Post 48 h transfection, WCEs were prepared and resolved on 10% SDS-PAGE followed by immunoblotting with C/EBPα, E6AP and β actin antibodies; lysates of 293T alone and transfected with C/EBPα were used as positive and negative control, note that there is no endogenous expression of C/EBPα in 293T. E6AP promotes C/EBPα degradation through ubiquitin-proteasome pathway: (c) 3T3-L1 preadipocytes pre-treated with MDI for 48 h were transiently transfected with expression plasmids for E6AP, E6AP-C843A and His-ubiquitin. Post 48 h Transfection, WCEs were prepared and C/EBPα was co-immunoprecipitated. IgG was used as a control. Co-immunoprecipitates were resolved on 8% SDS-PAGE and blots were probed with anti-His and anti-C/EBPα antibody respectively. E6AP inhibits C/EBPα transactivation potential to activate PPRE-Luc: (d) E6AP mediated downregulation of C/EBPα curtails its transactivation potential: 3T3-L1 preadipocytess were transiently transfected with pPPRE-luc reporter and expression plasmids for C/EBPα, E6AP and E6AP-C843A. 24 h post transfection, luciferase activity was measured. MG132 and lactacytin (LCN) treatment was given 3 h prior to cell harvesting for luciferase activity measurement. Data are representative of three independent experiments. Results are given as standard error of mean (± s.e.m.); *p<0.05; **p<0.001, ***<0.0001.