Acidic and cysteine residues within the Tat activation domain are required for interaction with PRMT6. (A) Schematic representation of the Tat activation domain from amino acids 1 to 37 and the basic domain from amino acids 49 to 57. The wild type (WT) amino acid sequence is shown using the single-letter amino acid code. Sequences of the acidic residues mutant (EDE), the cysteine residues mutant (CS) and the basic domain mutant (NB) are also shown, with substitution mutations indicated in reverse video. ARM, arginine rich motif. (B) Interactions between Myc epitope-tagged PRMT6 (Myc-PRMT6) and wild type, EDE mutant or CS mutant FLAG epitope-tagged Tat (Tat-FLAG) as determined by immunoprecipitation. HeLa cells were transfected to express Myc-PRMT6 with either wild type Tat-FLAG (WT) or one of its mutants as indicated. Immunoprecipitations were performed on lysates prepared from transfected cells using anti-FLAG agarose beads (αFLAG IP). Cell lysates (left panel) and immunoprecipitates (right panel) were western blotted using anti-Myc and anti-FLAG antibodies. (C) The same experiment as in B was performed with the NB mutant of Tat-FLAG. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. All data are representative of three independent experiments.