FIGURE 4.

EWSR1 and RNA Pol II are assembled in large protein assemblies. (A) Total protein from HEK293T/17 cell lysates was analyzed by SEC to separate proteins, complexes, or assemblies by size. The averaged ELISA signals are shown normalized to their maximum values for EWSR1 (C-9 antibody), yellow, or RNA Pol II (CTD4H8 antibody), red, as measured by ELISA. For samples from cells not crosslinked, top, RNA Pol II signals were highest in fractions eluting just before 20 mL, corresponding to particles of 25 nm in size. EWSR1 eluted after 20 mL or as a particle <25 nm in size. For crosslinked samples, bottom, the elution of EWSR1 and RNA Pol II shifted to early fractions with particles measured to be up to 150 nm in diameter. Dashed lines represent standard error about the mean (n = 3). (B) IP assays using cells without crosslinking enrich for ordinary and stable molecular complexes. Those using crosslinked cells can recover large molecular assemblies that form by weak protein interactions. (C, left) ELISA assays detected EWSR1 eluted from crosslinked IP assays of phosphorylated (S5P) RNA Pol II (Abcam, ab5131) in HEK293T/17 cells (n = 3). (Right) S5P RNA Pol II was detected for crosslinked IP of EWSR1 (B-1 antibody) (n = 3). (D, left) ELISA assays in A673 cells also detected EWSR1 eluted with S5P RNA Pol II. (Right) S5P RNA Pol II was detected in crosslinked IP assays of EWSR1 (n = 3). All error bars represent standard error about the mean. (AU) Absorbance units, (RLU) relative luminescence units. Student's t-test was calculated assuming equal variances: (**) P < 0.01; (*) P < 0.05; n.s., not significant (P > 0.05).