Fig. 4.

NHERF1 or NHERF2 knockdown individually in Caco-2/bbe cells did not change the cAMP inhibition of NHE3, but the cAMP effect was abolished when both NHERF1 and NHERF2 were knocked down. A: Caco-2/bbe cells stably expressing lentivirus-shRNA for GFP (control cell), NHERF1 (construct 1-1) and NHERF2 (construct 2-3) were grown on filterslips and infected with Adeno-HA-NHE3. NHE3 activity was measured in these cells with or without 20 μM forskolin (FSK) treatment for 15 min. FSK inhibited NHE3 activity (30–40%) in control cells, and similar inhibition occurred in NHERF1-KD and NHERF2-KD cells. Results are means ± SE from 3 independent experiments; P values represent effect of FSK (paired t-tests). B: Caco-2/bbe cells with NHERF2 knocked down (shRNA construct 2-3) were transduced with additional lentivirus prepared from shRNA for NHERF1 (construct 1-1), and cells were selected with a higher concentration of puromycin (20 μg/ml). After 2 passages, cell lysates were prepared and 40 μg of protein was used for Western blot analysis for NHERF1 and NHERF2 expression. In these double-knockdown cells, both NHERF1 and NHERF2 were effectively knocked down (>80%) compared with lentivirus-empty vector + shRNA GFP knockdown cells (control). C: NHE3 activity was measured in cells in which both NHERF1 and NHERF2 were knocked down with lentiviruses, with or without 20 μM FSK treatment for 15 min. FSK inhibited NHE3 activity in control cells (lentivirus empty virus + shRNA GFP), but the FSK effect was abolished when NHERF1 + NHERF2 were both knocked down. Results are means ± SE of 3 separate experiments. P values compare effects of FSK and control, with control being a combination of lentivirus-GFP-shRNA and empty vector (paired t-tests).