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. 2010 Dec 29;300(4):C771–C782. doi: 10.1152/ajpcell.00119.2010

Fig. 3.

Fig. 3.

Stable knockdown of NHERF1 or NHERF2 in Caco-2/bbe cells using lentivirus-short hairpin RNA (shRNA) to study NHE3 regulation. A: Caco-2/bbe cells were transduced with lentiviral particles prepared from 3 different shRNA constructs specific for either NHERF1 or NHERF2 genes. After selection with puromycin (10 μg) for at least 2 passages, cell lysates were prepared; 40 μg of cell lysate was used to immunoblot with anti-NHERF1 and -NHERF2 antibodies. Cells infected with lentivirus prepared from shRNA specific for green fluorescent protein (GFP) was used as a negative control (Cont; human cells do not endogenously express GFP). NHERF1 shRNA construct 1-1 knocked down >90% NHERF1 expression, and NHERF2 shRNA construct 2-3 knocked down NHERF2 expression by >85%. Intensities of protein expression were calculated with Odyssey software. Results shown are from a single experiment that was repeated 4 times with similar results. B: Caco-2/bbe cells grown on filterslips were infected with Adeno-NHE3, and NHE3 basal activity was measured with BCECF/fluorometry. Cells with NHERF1 knocked down (shRNA construct 1-1) had significantly reduced NHE3 activity (30–40%) compared with wild-type (WT) or GFP-knockdown (KD) controls. Cells with NHERF2 knocked down (shRNA construct 2-3) had significantly increased NHE3 activity (6). Results represent means ± SE from n independent experiments. Control was GFP-shRNA (6). shRNA for GFP did not alter basal NHE3 activity compared with empty vector (data not shown). P values are comparison with control (ANOVA). C–E: apical cell membrane biotinylation was performed in Caco-2/bbe cells transduced with lentivirus-shRNA-GFP, shRNA-NHERF1, and shRNA-NHERF2. Total and apical surface amounts of NHE3 expression were determined by Western blot analysis. Two different volumes of each sample (10 μl and 20 μl) were loaded for Western blot analysis. Total and surface NHE3 expression were normalized to total β-actin. Calculated total and surface NHE3 used as a control lentivirus-shRNA-GFP set to 100% for each experiment. Results are means ± SE of 4 independent experiments. P values are in comparison to control (unpaired t-tests). NS, not significant.