FIG. 5.
The purified protein which forms complexes 1 and 2 is supershifted by an anti-HuR antibody. A sample of fraction 8 from the Mono-S column (as in Fig. 3) was mixed with 3.5 μg of anti-HuR antibody or murine immunoglobulin G and incubated for 10 min at RT. A 20-fmol portion of the 75-nt probe was added, and the sample was incubated for 1 h on ice. RNase T1 and heparin sulfate were added, and the sample was incubated for 5 min at RT. Samples were then electrophoresed as for EMSA. A high concentration (1:2 dilution [left panel]) and a lower concentration (1:4 dilution [right panel]) of fraction 8 were used.