Figure 10.
CaM has reduced binding to NHE3 Δ4 but CaM and SNX27 have normal binding to NHE3 PBMIImut. A, HA-tagged NHE3 constructs were immobilized by monoclonal anti-HA affinity matrix and co-precipitated with lysates of PS120, NHE3 WT, NHE3 PBMIImut, and NHE3 Δ4 in PS120/NHERF2 cells. CaM had reduced binding to NHE3 Δ4 but bound like NHE3 WT to NHE3 PBMIImut. Samples were detected by Western blotting (IB) with monoclonal anti-CaM antibody (Sigma) and monoclonal HA antibody (Sigma). A representative experiment is show, and these experiments were repeated three times with similar results. B, NHE3 PBMIImut bound SNX27 like NHE3 WT. We previously reported that NHE3 Δ4 had minimal SNX27 binding (32). GST or GST-tagged SNX27 was immobilized by GSH beads used to perform in vitro pulldown assays with the HA-tagged NHE3 WT and NHE3 PBMIImut. Samples were analyzed by Western blotting (IB) with antibodies against GST (Cell Signaling Technology) and HA (Sigma).