Figure 2.

Depletion of HuR decreases Cx43 levels and Cx43 mRNA stability. (A) WB-F344 cells were treated with HuR-specific or control siRNA for 48 h, followed by Real-time RT-PCR analysis of HuR and Cx43 mRNA levels. Data were normalized against 18S rRNA levels and control treatments were set to 1. Data are means of two independent experiments. (B) Western blot analysis of Cx43 levels in WB-F344 cells treated with control or HuR-specific siRNA. Depletion of HuR and loading of gels were controlled for by Western analysis of HuR and GAPDH levels. Data are means ± SEM (n=3). (C) WB-F344 cells were transfected with control or HuR-specific siRNA for 30 h, followed by exposure to actinomycin D for the indicated periods of time, lysis of cells and analysis of Cx43 mRNA levels by RT-PCR. Changes in mRNA levels were assessed by densitometric analysis of ethidium bromide-stained PCR product bands in agarose gels and normalized against GAPDH mRNA. Normalized Cx43 mRNA levels prior to addition of actinomycin D were set to 100%. Data are means of two to three independent experiments ± SEM. (D) Cells were treated with actinomycin D as in (C), followed by Real-time quantitative RT-PCR analysis of Cx43 and GAPDH mRNA levels in cells treated with control or HuR-specific siRNA. Data were normalized to 18S rRNA levels and are means of two independent experiments.