Fig. (5) Silencing of endogenous HuR reduces expression of HDAgs in Huh7-D12 cells. (A) Knockdown of the HuR protein was performed using a specific siRNA in Huh7-D12 cells. 48 hrs after addition of puromycin, total protein extracts were prepared, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Blots were incubated with anti-HuR, anti-clathrin or anti-HDAg antibodies, and the displayed images are representative of three independent experiments. (B) Images were digitalized and band intensity was determined using the ImageJ program. Relative expression levels were obtained after normalization with the housekeeping protein clathrin. Inhibition of HDAg expression is presented as a percentage after comparison with the values obtained with the negative control vector pSIREN-RetroQ/Luc provided by the manufaturer.