Fig. 1

Characterization of AFF4-associated proteins. a Schematic domain organization of human AFF4 and FUS proteins. AFF4 serves as a scaffold for the assembly of SEC subunits including the elongation transcription complexes ELL1/2 and P-TEFb [83]. At its N-terminal region it consists the P-TEFb binding domain (orange); the ELL domain (yellow); ENL/AF9 (black); and a C-terminal motif (orange). In FUS, the N-terminal of the protein exhibits low sequence complexity (blue). The RGG domains contain a triplet repeat motif of arginine–glycine–glycine (green). FUS also contains an RNA-recognition motif (RRM) (red), a zinc-finger domain (ZnF) gray), and a proline–tyrosine nuclear localization signal (PY–NLS; dark red) [55]. b Purification of AFF4 protein partners by affinity purification. Control HEK293T cells, or cells expressing either Full length HA-AFF4 (1–1163) or its truncated form HA-AFF4 (1–300) were subjected to HA-epitope-tagged immuno-purification (IP) with anti-HA antibody. IP samples were separated by SDS-PAGE and visualized by silver staining. Cellular partners of HA-AFF4 proteins were also identified by tandem mass spectrometry. Previously confirmed SEC/P-TEFb partners as well as newly identified partners like FUS were recovered by our MS analysis (also see Additional file 1: Figure S1, Additional file 2: Table S1 and Additional file 3: Table S2 for quantitative analysis of the MS results). Red asterisks indicate potential novel AFF4 partners, which FUS is one of them: Blue asterisks point to the full-length and truncated AFF4 (1–300) proteins. c N-terminus region of AFF4 associates with FUS in cells. Western blot analysis of immuno-precipitation (IP) samples defining the regions of AFF4 that mediate association with the FUS in cells. Lysates from HEK293T cells expressing Flag-FUS and the indicated HA-AFF4 proteins were IP with anti-HA IgG. IP. IP and input (5%) samples were analyzed by SDS-PAGE followed by western blot analysis with anti-HA or anti Flag antibodies