Fig. 2.

K999, K1034, and K1057 are the SUMO acceptor sites in CBP. COS-1 cells transfected with expression construct of Flag-tagged CBP5 WT and various KR mutants (A) or Flag-tagged CBP WT and 3KR mutants (B) along with or without EGFP-SUMO-1 as indicated were lysed in RIPA buffer with 10 mM NEM followed by immunoprecipitation and Western analysis with indicated antibodies. A scheme indicating the migrating position of the single and double EGFP-SUMO-1-conjugated CBP5 fragments are shown in Fig. 8, which is published as supporting information on the PNAS web site. The arrowhead depicts the SUMO-1-modified CBP fragments (A) or full-length CBP (B). The arrow and asterisk indicate CBP fragments and nonspecific bands, respectively. The open arrowhead indicates the position of sumoylated CBP species is missing because of mutation.