Fig. 2
AAG directly interacts with ELP1 subunit of transcriptional Elongator complex. a Silver staining analysis of untreated or MMS (methyl methanesulfonate) treated FLAG-AAG immunoprecipitation (IP) samples subjected to LC/MS-MS analysis. IP sample from HEK293T cells transfected with empty FLAG vector served as negative control. LC/MS-MS analysis of FLAG-AAG IP samples identified ELP1 as the most specifically enriched AAG interacting partner. Remaining subunits of core Elongator (ELP2 and 3), and known interacting partner PCNA are depicted. See also Supplementary Data 2 for the complete list of proteins identified in all samples. b IP of AAG from HEK293T whole cell extracts (WCEs) treated or untreated with MMS. c Immunoblot analysis of gel shift experiments with purified recombinant AAG and FLAG-ELP1 proteins, and chemical crosslinking with indicated amounts of BS3 (bissulfosuccinimidyl suberate). Control samples – proteins in the absence of crosslinking agent (lanes 1-3); and single proteins with highest amount of BS3 (lanes 6, 7). d Schematic representation of full-length AAG, AAG lacking 80 N-terminal amino acids (∆80 AAG), and first 80 N-terminal amino acids of AAG (1-80aa AAG); numbers indicate amino acids. e Co-IP of purified FLAG-ELP1 with AAG full length, or AAG lacking 80 N-terminal amino acids (∆80 AAG). f IP of GFP-tagged first 80 N-terminal amino acids of AAG (1-80aa AAG) (GFP-1-80aa AAG) expressed in HEK293T AAG−/− cells. GFP-tag positioned N-terminally. g Proximity ligation assay (PLA) showing the AAG-ELP1 interaction in HEK293T cells. Scale bar: 50 μm. Source data are provided as a Source Data file.
