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. 2017 Jul 1;28(13):1754–1767. doi: 10.1091/mbc.E16-12-0862

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© 2017 Sarker et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — NHERF2 and NHERF3 bind to NHE3-WT and NHE3-S719D normally but bind less to NHE3-S719A. The NHERF2 coIP of NHERF3 is similarly reduced in NHE3-S719A cells. (A) IP of FLAG-NHERF2 was performed using cell lysates prepared from Caco-2/bbe cells grown in 10-cm Transwell plates also infected with Ad-HA-NHE3-WT, -S719A, or- S719D and Ad-FLAG-NHERF2. IP FLAG–magnetic beads were washed, and bound proteins were eluted and separated by SDS–PAGE. Proteins were transferred to membrane and immunoblotted with anti-HA (monoclonal), anti-NHERF3 (polyclonal), anti-ezrin (polyclonal), and anti-FLAG (monoclonal) antibodies. Protein bands were visualized and quantitated with the Odyssey system and LI-COR software for the IR-Dye secondary antibodies. (B) NHE3, NHERF3, and NHERF2 protein bands from experiments as in A were quantitated with the Odyssey system and LI-COR software. Coimmunoprecipitated protein amount was normalized with immunoprecipitated protein amount (FLAG-NHERF2) and expressed as percentage of WT proteins. NHE3-S719A has greatly reduced binding to NHERF2 compared with WT and S719D. NHERF3 coIP by NHERF2 was also reduced in NHE3-S719A but normal in NHE3-S719D cells. (C, D) Caco-2/bbe/Lenti-NHERF3 knockdown cells were infected with Ad-HA-NHE3-WT, -S719A, or -S719D and Ad-FLAG-rat-NHERF3 (Lenti-NHERF3 short hairpin RNA has no effect on rat FLAG-NHERF3 expression). NHE3-WT and S719D showed similar binding with NHERF3, but NHE3-S719A had significantly less binding. n, number of experiments. p values are by unpaired t tests and ANOVA. (E) IPs of ezrin (mAb), FLAG (mAb), and immunoglobulin G (mAb) were performed using cell lysate prepared from Caco-2/bbe cells grown in 10-cm Transwells plates and infected with Ad-HA-NHE3-WT or -S719A and Ad-FLAG-NHERF2. Immunoblotting was with anti-HA, anti-ezrin, anti-NHERF3, and anti-FLAG antibodies. Binding of ezrin with NHE3-S719A was drastically reduced compared with NHE3-WT.