Figure 7

Affinity-enrichment analyses for ZMRE-binding factors. (A) SDS-PAGE of retentate from ζWT, ζ57, and ζ65 ssDNA probes, and from control agarose beads. Bands corresponding to two AUF1 isotypes are indicated with an asterisk. M = molecular size marker. (B) Confirmatory immunoblot analyses. Retentate was assessed using antibodies against AUF1, RPA70 (positive control, binds all three probes), and hnRNP H (negative control, present in input nuclear and cytoplasmic fractions). Similar results were obtained in parallel analyses of extracts from HeLa cells (not shown). (C) RNA immunoprecipitation analyses. Experiments were conducted on extract from HeLa cells that express ζ-globin mRNA, using AUF1 antibody or control polyspecific immunoglobulin (Ig) control. c-Myc mRNA was assessed in parallel as a positive control for AUF1 binding. M = molecular size marker; C = water-only RT-PCR control; I = control input (unprecipitated) mRNA.