Fig 1: Quantitative proteomic analysis of FUS/TLS using stable isotope labeling by amino acids in cell culture (SILAC) coupled with tandem affinity purification (TAP)(a) Schematic representation of FST/TLS tagging including the localization and affinity purification (LAP)-tag composed of GFP followed by PreScission protease cleavage sequences, a 6x histidine-tag, and an HA-peptide (YPYDVPDYA). (b) Immunoblotting of FUS/TLS showed comparable levels of cell extracts for GFP-tagged or endogenous FUS/TLS. (c) GFP fluorescence of induced GFP-tagged FUS/TLS and immunofluorescence of endogenous FUS/TLS on HeLa Flp-In cells. (d) Schematic representation of TAP purification and quantitative analysis using SILAC. (e-f) Silver stain of the samples from the PreScission (e) and final (f) elutions of the tandem affinity purification steps. Arrow indicates tagged FUS/TLS and * denoted GST-tagged PreScission protease; positions of other visible polypeptides are marked with dashes. (g) Graphic representation of FUS/TLS-interactome. Y-axis displays the average ratio of different peptides per identified protein; the X-axis displays protein ID in alphabetical order, with a fourfold enrichment above background signal considered to be an FUS/TLS-associated protein (highlighted in colors that match identities in (h)). (h) Summary of FUS/TLS associated proteins, identified with peptides containing only heavy Lys/Arg and by multiple different peptides from at least two independent runs, except *, which denotes the proteins were identified only in one run. The associated proteins were grouped according to the known assigned functions for each. (i) Confirmation of putative FUS/TLS associating proteins by immunoblotting, including U1-70K, U1-A, U1-C, Sm-B/D, CPSF-160, PABPC1, GEMIN5, SMN.