Characterization of HIV Tat modifications using novel methyl-lysine-specific antibodies.
Modification-specific antibodies are important tools to examine the dynamics and functions of posttranslational protein modifications in cells. Here, we describe in detail the generation of polyclonal antibodies specific for mono-, di-, and trimethylated lysine 51 within the HIV transactivator Tat. Lysine 51 is a highly conserved residue located in the RNA-binding region of Tat and the target of lysine methyltransferases KMT1E (SETDB1) and KMT7 (Set7/9). Using affinity-purified methyl-specific antibodies of Tat, we find that cellular Tat is predominantly monomethylated at lysine 51, a modification enhanced by coexpression of KMT7.
Pagans
Sara
S
Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94158, USA.
Sakane
Naoki
N
Schnölzer
Martina
M
Ott
Melanie
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D057809:HEK293 Cells; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D008745:Methylation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D054322:tat Gene Products, Human Immunodeficiency Virus; C005205:epsilon-N-methyllysine; D008239:Lysine