TY - JOUR
T1 - Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin
AU - Wang, Haopeng
AU - Matsuzawa, Atsushi
AU - Brown, Scott A.
AU - Zhou, Jingran
AU - Guy, Cliff S.
AU - Tseng, Ping Hui
AU - Forbes, Karen
AU - Nicholson, Thomas P.
AU - Sheppard, Paul W.
AU - Häcker, Hans
AU - Karin, Michael
AU - Vignali, Dario A.A.
PY - 2008/12/23
Y1 - 2008/12/23
N2 - Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.
AB - Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.
KW - MAP kinase kinase kinase 1 (MEKK1)
KW - TNF receptor-associated factor 6 (TRAF6)
UR - http://www.scopus.com/inward/record.url?scp=58149520089&partnerID=8YFLogxK
U2 - 10.1073/pnas.0810461105
DO - 10.1073/pnas.0810461105
M3 - Article
C2 - 19091944
AN - SCOPUS:58149520089
SN - 0027-8424
VL - 105
SP - 20197
EP - 20202
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 51
ER -