Skip to main content
. 2017 Mar 10;292(20):8279–8290. doi: 10.1074/jbc.M116.774489

Figure 3.

Figure 3.

NHERF2 co-precipitates NHE3 WT but has reduced association with NHE3 PBMIImut, NHE3 Δ4, and NHE3 PBMIImut + Δ4. A, to compare the roles of internal PBM (-595CLDM-) and C-terminal putative Class I PBM (-829STHM), the HA-tagged NHE3 constructs NHE3 WT, NHE3 PBMIImut, NHE3 Δ4 (C-terminal four amino acids, STHM, deleted), and NHE3 PBMIImut + Δ4 were designed in pcDNA3.1_Hygro vector and stably transfected into PS120/NHERF2 cells. B, immunoprecipitation was performed with anti-NHERF2 antibody (α-2570) from PS120/HA-NHE3 WT (as a negative control; first lane), PS120/NHERF2/HA-NHE3 WT (second lane), PS120/NHERF2/HA-NHE3 PBMIImut (third lane), PS120/NHERF2/HA-NHE3 Δ4 (fourth lane), and PS120/NHERF2/HA-NHE3 PBMIImut + Δ4 (fifth lane). NHERF2 co-immunoprecipitated NHE3 WT but not the negative control (first lane) and immunoprecipitated reduced amounts of NHE3 PBMIImut, NHE3 Δ4, and NHE3 PBMIImut + Δ4. Input and bound NHE3 were detected with monoclonal anti-HA antibody. NHERF2 was detected by polyclonal anti-NHERF2 antibody (α-2570). A representative experiment of three similar experiments is shown above, and quantitation of the amount of NHE3 immunoprecipitated by NHERF2 normalized to the amount of NHERF2 immunoprecipitated is show below. Results are mean ± S.D., n = 3. Error bars represent S.D. Means are shown in this and subsequent figures as open symbols, whereas individual experiments are shown as filled symbols. p values compare results between mutants. C, pulldown assays were performed to determine the interactions between NHE3 (WT, PBMIImut, Δ4, and PBMIImut + Δ4) and NHERFs (NHERF1 (NF1), NHERF2 (NF2), and NHERF3 (NF3)). GST or GST-tagged NHERF1, NHERF2, and NHERF3 were used to pull down HA-tagged NHE3 WT, PBMIImut, Δ4, and PBMIImut + Δ4 in lysates from PS120/NHERF2 cells. The left panel shows the total cell lysates of GST and GST-NHERF1, -NHERF2, and -NHERF3 used for pulldown assays. The right panel shows the pulldown results. A representative experiment of three similar experiments is shown. NS, not significant; IB, immunoblotting.