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. 2001 Mar 27;98(8):4455–4460. doi: 10.1073/pnas.081061398

Figure 1.

Figure 1

p19ARF promotes E2F1 degradation. (A) p19ARF affects E2F1 abundance and electrophoretic mobility. pRclCMV-HA-E2F1 (1 μg), pCD-p19ARF (5 μg), or vector alone (5 μg) were cotransfected into U2OS cells. Cell extracts were prepared and examined by SDS/PAGE on 12% gels and Western blotting using antibody specific for E2F1 (C20, Santa Cruz Biotechnology). Endogenous E2F1 is not detected in this experiment. (B) Pulse–chase analysis of E2F1 in the absence (◊) or presence (□) of p19ARF. pRclCMV-HA-E2F1 (1 μg) and pCD-p19ARF (5 μg) or vector alone (5 μg) were cotransfected into U2OS cells. Cells were pulsed-labeled with [35S]methionine for 30 min and chased for 1 or 3 h, followed by immunoprecipitation with anti-E2F1 antibodies (C20). Under these conditions, endogenous E2F1 was not detected (data not shown). The amount of transfected E2F1 in specific bands (as detected with a PhosphorImager) was plotted as a function of time. (The E2F1 signal detected at time 0 was set at 100% and used to normalize the signal at the subsequent times.) (C and D) E2F2 and E2F3 are targeted for degradation by p19ARF. pRclCMV-HAE2F2 or -3 expression plasmids (2 μg) were transfected into 293T cells with an empty vector (5 μg) or pCD-p19ARF (5 μg), and cell lysates were analyzed for ectopic E2F2 or -3 mRNA and protein levels. Lysates were analyzed by Western blotting for HA (F-7, Santa Cruz Biotechnology) and for p19ARF (AEC40), and gel loading of each lysate was normalized for E2F2 or -3 mRNA levels. The upper band in both lanes of C and D is nonspecific. (E) E2F6 is not targeted for degradation by ARF. pCDNA3-HAE2F6 (2 μg) or pRclCMV-HAE2F1 (2 μg,-a positive control for ARF-induced degradation) were cotransfected with empty vector (5 μg) or pCD-p19ARF(5 μg) into 293T cells, and lysates analyzed for ectopic E2F6 and E2F1 protein and mRNA levels. Lysates were analyzed by Western blotting for HA (F-7) and p19ARF (R562, Abcam, Cambridge, U.K.), and gel loading of each lysate was normalized for E2F6 or E2F1 mRNA levels, respectively. (F) LLnL suppresses p19ARF-mediated destabilization of E2F1. pRclCMV-HAE2F1 (2 μg) was cotransfected with control vector (5 μg) or pCD-p19ARF (5 μg) into U2OS (Upper) or 293T cells (Lower). Twenty-four hours after transfection, plates were incubated in 50 μM LLnL/0.1% DMSO or 0.1% DMSO for 12 h, and ectopically expressed E2F1 mRNA levels were measured in the transfected cells by Northern blotting. Cell lysates from the same cultures were loaded onto an SDS gel with loading normalized for the E2F1 mRNA levels present in the cells of origin. HA-E2F1 and p19ARF levels were determined by Western blotting with 12CA5 (Roche) anti-HA and AEC40 antibodies, respectively.