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.