p300 and CBP co-immunoprecipitate with REL and act as REL coactivators. (A) Anti-FLAG immunoprecipitation (IP: FLAG) was performed on nuclear extracts of A293 cells that had been transfected with 5 μg of expression plasmids for the indicated FLAG-REL fusion proteins and 5 μg of HA-p300. Immunoprecipitates were subjected to anti-HA Western blotting (to detect p300; top panel). For each sample, 1% of the amount of extract used for one IP (5 μg) was included on the gel an as input lane (left 5 lanes). Anti-FLAG Western blotting (WB: FLAG) of 1% of each extract shows approximately equal expression of each FLAG-REL fusion protein (lower panel). * - indicates a nonspecific band. (B) Anti-MYC immunoprecipitation (IP: MYC) was performed on nuclear extracts of A293 cells that had been transfected with 5 μg of the indicated MYC-REL fusions and 5 μg of pCMV-CBP. Immunoprecipitates were subjected to anti-CBP Western blotting (top panel). One percent of the extract used in each immunoprecipitation was included as an input for each sample (left 3 lanes). Anti-MYC Western blotting of 1% of each extract shows approximately equal expression of each MYC-REL fusion protein (lower panel). (C) p300 and CBP enhance REL-mediated transcriptional activation. A293 cells were transfected with 0.5 μg of a 3x-κB-site luciferase reporter plasmid and 0.5 μg of normalization plasmid RSV-βgal. Cells were co-transfected with 0.5 μg of pcDNA-REL or vector alone; and 0.5 μg of HA-p300, or vector alone (left panel). A similar experiment was performed using pCMV-CBP in place of HA-p300 (right panel). Luciferase and β-galactosidase activites were determined, and values were normalized as indicated (1.0). Values are the averages of three experiments performed with triplicate samples.

p300ΔC interacts with REL in RC-K8 cells and with REL TAD sequences in vitro. (A) RC-K8 (top) and REL-transformed chicken spleen (bottom) cell nuclear extracts were immunoprecipitated (IP) with normal rabbit IgG or anti-p300 antiserum. Immunoprecipitates were subjected to anti-REL or anti-p300 Western blotting, as indicated to the side of each panel. One percent (2.5 μg) of the nuclear extract used for immunoprecipitation was included as an input for the anti-REL blots and 10% of the nuclear extract (25 μg) was used as input for the anti-p300 blots. (B) RC-K8 cell nuclear extracts were immunoprecipitated with normal rabbit IgG or anti-CBP antiserum. As in (A), immunoprecipitates were subjected to anti-REL (top panel) or anti-CBP (lower panel) Western blotting with input controls. (C) GST-REL pulldown of p300ΔC from RC-K8 whole cell extract. Bound proteins were subjected to anti-p300 Western blotting (top panel). Coomassie Blue staining was performed on 5% of GST or GST-fusion proteins used in the pulldowns (lower panel).

EP300 sequences are deleted after exon 17 on one allele in RC-K8 cells. (A) Real-time PCR for individual exons of EP300 was performed using BJAB and RC-K8 genomic DNA. Specific primer sets for exons 2, 13–25 and 27 were used for PCR amplification of genomic DNA. Values were normalized to those of EP300 exon 2 (ΔC_t), and then to those of BJAB DNA (Δ ΔC_t) (1.0). (Exon 26 was omitted because it was difficult to design appropriate primers for this short exon.) (B) Schematic diagram showing the structure of human p300. Residues 1224–1669 constitute the histone acetyltransferase (HAT) domain and the N- and C-terminal regions of p300 form a transactivation domain. The arrow indicates the end of exon 17-encoded residues. It is predicted that p300ΔC contains only those residues encoded by exons 1–17; as such, p300ΔC would lack the bromodomain, the HAT domain, and the C-terminal transactivation domain. CH1-3, cysteine/histidine-rich domains; KIX, CREB-binding domain; BD, bromodomain; QP, glutamine/proline-rich.

CBP and p300 interact with REL transactivation domain sequences in vitro. (A) Structures of full-length REL (top) and GST-REL fusion proteins used in this study. RHD, Rel homology domain; TAD, transactivation domain; GST, glutathione-S-transferase. (B and C) GST-REL pulldown of endogenous CBP from A293 whole cell extracts. Bound proteins were subjected to anti-CBP Western blotting (top panels). One percent of the amount of extract used for one pulldown (30 μg) was included on the gel as an input lane. (D) GST-REL pulldown of endogenous p300 from A293 whole cell extracts. Bound proteins were subjected to anti-p300 Western blotting (top panel). For each GST pulldown, Coomassie Blue staining was performed on 5% of GST or GST-fusion protein used in the pulldowns (lower panels).

RC-K8 cells express a C-terminally truncated p300 protein. (A) Anti-p300 Western blotting of A293 and RC-K8 whole cell extracts. Twenty-five μg of A293 extract and 25, 50, 100, and 200 μg of RC-K8 extract were loaded. (B) The six indicated B-lymphoma cell lines, A293 cells, and REL-transformed chicken spleen cells (REL Spl.) were analyzed by Western blotting for expression of p300 and CBP. Fifty μg of whole cell extract was loaded, except for RC-K8 extracts, where 50 and 100 μg were loaded. Western blots were performed with N-terminal (top panel) and C-terminal (2nd panel from top) anti-p300 antisera and with anti-CBP antiserum (3rd panel from top). Coomassie Blue staining shows approximately equal loading of all extracts (bottom panel). (C) RC-K8 cells express a truncated REL protein called REL-NRG and no IκBα Forty μg of A293 and RC-K8 whole cell extract was loaded and anti-REL and anti- IκBα Western blotting was performed. Anti-β-tubulin Western blotting was performed as a loading control.

RC-K8 cells express a 3′ truncated EP300 mRNA. (A) Northern blotting using probes from 5′ (exons 1–2) and 3′ (exon 31) sequences of EP300 was performed. Fifty, 50, and 20 μg of A293 RNA and 80, 50, and 20 μg of RC-K8 RNA were loaded. The positions of the 28S (~4.7 kb) and 18S (~1.9 kb) rRNAs, as determined by methylene blue staining of the filter, are indicated to the side of each Northern blot. (B) Real-time PCR on EP300 was performed using A293 and RC-K8 cDNA. Specific primer sets for sequences encoded by exons 2 and 31 of EP300 and for GAPDH were then used for PCR amplification. To obtain relative EP300 mRNA levels, values were normalized to GAPDH values (ΔC_t) and then to those of A293 cDNA (ΔΔC_t) (1.0). (C) Semi-quantitative RT-PCR on EP300 was performed using A293 and RC-K8 cDNA. Specific primer sets were used to amplify sequences encoded by exons 3–12 and exon 31 of EP300. Band densitometry was measured using ImageJ software. Values were normalized to those of A293 cDNA (1.0). NTC denotes a no template control.