Figure 7. EGR-1 siRNA suppresses the PMA-induced hNHE3 mRNA up-regulation.

C2BBe1 cells were transfected with a control siRNA or EGR-1-specific siRNA and treated with PMA as described in the Experimental section. Total cell proteins (15 μg/lane) were resolved in an SDS/10% polyacrylamide gel, subsequently transferred on to Immobilon-P and sequentially probed with antibodies specific to EGR-1 (A) and tubulin (B) as a control for the protein loading. The effect of EGR-1 down-regulation on PMA-induced NHE3 mRNA expression was determined by RT–PCR (C). Total RNA (3 μg) from the cells treated as above was subjected to reverse transcription using oligo(dT) as primer. One-tenth of the reverse transcription product was PCR-amplified as described in the Experimental section using NHE3-specific primers. Total proteins and RNA from untransfected cells were also used as a control in Western blotting and RT–PCR analysis.