Fig. 2.
Detection of HuR. (A) 2D plot of RNA pull-down result for HDAC2 mRNA 3′ UTR. The log2 SILAC ratio of proteins identified with at least 2 unique peptides in each MS run is plotted of the forward pull-down (x axis) against the cross-over pull-down (y axis). Specific interaction partners show inverse ratios between forward and reverse experiment, grouping them into the upper left quadrant. The interval for statistical significance (shaded in the plot) is given by the product of significance in the forward and reverse experiment (P(for) × P(rev) < 0.0001) and a minimum SILAC ratio change of a factor 2 (expected experimental variation). Among 552 identified proteins before filtering, the known interaction partner for the HDAC2 3′ UTR fragment (HuR) is represented by a black bullet, having the highest SILAC ratio in the forward and smallest in the cross-over experiment. (B) Western blot confirmation of the MS analysis for HuR. (C) RIP of in vivo cross-linked RNP complexes with antibodies against HUR, MATR3 and isotype control shows enrichment of HDAC2 mRNA at the identified candidates. (D) Differential binding of HuR can be detected by comparing MTA1 3′ UTR wild-type sequence with a deletion variant (SILAC ratio 11.8) or a mutated variant with a destabilized stem loop structure (SILAC ratio 6.5). Between the wild-type sequence and a point mutated variant, no differential binding (SILAC ratio 1:1) is observed. (E) Results of quantitative MS are supported by Western blot analysis. HuR binds to wild-type sequence and the point mutated variant U15G.