Figure 7

Effect of apocynin on HuR binding to CCL2 RNA. A,HuR expression was monitored by Western blot under various treatment conditions: unstimulated, treatment of cells with TNF-α or TNF-α/apo for 24 h. Data is a representative blot of n = 3. B, Quantitation of HuR protein level after various treatments relative to unstimulated cells by densitometry, using β-tubulin as a loading control for normalization. C,binding of HuR to CCL2 3'UTR by biotin affinity isolation. Lysates were incubated with biotinyated CCL2 3'UTR RNA, mixed with streptavidin beads, washed, and isolated proteins were analyzed by Western blot to identify HuR binding. TNF,Apo', cells were treated with TNF-α for 24 hours, lysed and then treated with apocynin (1 mM) for 30 min. Blot of representative data of n = 4. Each lane was taken from the same exposure of the same Western blot and re-arranged in order for the figure (L- pure lysate, PD- lysate after treatment with beads and biotinylated RNA). D, Densitometeric analysis of the Western blot data. (*, p < 0.05 when compared to TNF:No Tx Ratio), n = 4).