FIG. 2.
The Rb1ΔL-encoded protein fails to interact with chromatin-regulating factors and is defective for transcriptional repression. (A) pRb, p107, and p130 protein expression levels were measured in wild-type, Rb1ΔL/ΔL, and Rb1−/− MEFs by Western blotting (top), and the ability of GST-E7 to interact with pocket proteins was tested in GST pull-down assays and detected by Western blotting (bottom). The asterisk denotes a cross-reactive band in p130 blots. (B) The ability of pRbΔL to interact with E2F transcription factors was detected by electrophoretic mobility shift assay. Extracts from wild-type, Rb1ΔL/ΔL, and Rb1−/− cells were incubated with a radiolabeled double-stranded DNA oligonucleotide containing a consensus E2F binding site. Gel shift complexes were competed with unlabeled wild-type and mutant oligonucleotides (lanes 3, 4, 8, 9, 13, and 14). Complexes containing pRb and E2Fs were identified by supershifting with an α-pRb monoclonal antibody (lanes 6, 11, and 16). (C) The identities of proteins coisolated with GST-Rb from nuclear extracts in an LXCXE binding cleft-dependent manner were determined by Western blotting. Nuclear extract is abbreviated as NE. (D) GST-Suv4-20h1 and -2 proteins were used to coprecipitate pRb from wild-type, Rb1ΔL/ΔL, and Rb1−/− extracts. Western blots of input and precipitated protein levels are shown. An asterisk denotes background caused by the high-molecular-weight GST-Suv4-20h1 protein. (E) Northern blots were performed on RNA extracted from serum-starved or proliferating MEFs to assess expression of E2F target genes. The blots were probed to quantify message levels for p107, Cyclin E1, Thymidylate synthase, Cyclin E2, Cyclin A2, and ARPP0 (loading control). Asynch, asynchronous.