EBER1 binds predominantly the p40 isoform of AUF1 in vivo. (A) Experimental outline for UV crosslinking and coimmunoprecipitation of EBER1 to detect in vivo interactions. (B) The four Flag-tagged isoforms of AUF1 were transiently expressed individually, along with EBER1, in HEK293T cells. Immunoprecipitation using anti-Flag antibodies was carried out either omitting or including UV crosslinking, followed by RNA isolation and detection of EBER1 by Northern blot. All AUF1 isoforms bind EBER1 in vivo, with the p40 isoform showing the strongest crosslinking efficiency. Anti-Flag antibodies were used in a Western blot to verify the expression of each AUF1 isoform. (C) Competition experiments were carried out, showing that the enhanced interaction of p40AUF1 with EBER1 is due to preferential binding rather than preferential crosslinking efficiency. Flag-p40AUF1 and EBER1 were coexpressed in HEK293T cells with either an empty vector, Myc-tagged p40AUF1, or Myc-tagged p45AUF1, and immunoprecipitation was carried out using anti-Flag antibodies. The plasmids expressing Myc-tagged AUF1 were transfected in 10-fold molar excess over the Flag-tagged isoform. Western blots using anti-Flag or anti-Myc antibodies were carried out to show comparable expression of Flag-p40AUF1 or the Myc-tagged AUF1 isoforms. (D) In vivo UV crosslinking of Raji cells was followed by immunoprecipitation using anti-L22 (as a positive control) and anti-AUF1 antibodies, and EBER1 was detected by Northern blot analysis. The same blot was probed for EBER2 as a specificity control.
