Figure 2.

Mutation in putative Class I (-⁵⁹²SAV-) and Class II (-⁵⁹⁵CLDM-) internal PBMs and testing of their NHERF2 binding. A, one Class I PBM mutant, NHE3SAV/AA (⁵⁹²SAV → ⁵⁹²AAA), and two Class II PBM mutants, NHE3CLDM/EK (-⁵⁹⁵CLDM- → -⁵⁹⁵CEDK-) and NHE3CLDM/QA (-⁵⁹⁵CLDM- → -⁵⁹⁵CQDA-), were constructed. B, in vivo binding assays were performed. HA-tagged NHE3 constructs were immobilized by monoclonal anti-HA affinity matrix or by monoclonal anti-VSV-G antibodies immobilized on agarose as a negative control and co-precipitated with lysates from NHE3_WT, NHE3SAV/AA, NHE3CLDM/EK, and NHE3CLDM/QA in PS120/NHERF2 stable cell lines. NHE3_WT and NHE3SAV/AA precipitated NHERF2, but neither NHE3CLDM/EK nor NHE3CLDM/QA bound NHERF2. Monoclonal anti-HA antibody and polyclonal anti-NHERF2 antibody (α-2570) were used to detect HA-tagged NHE3 constructs and NHERF2, respectively. A representative experiment of three similar experiments is shown. IB, immunoblotting.