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Figure 1 | Breast Cancer Research

Figure 1

From: Na+/H+exchanger regulatory factor 1 inhibits platelet-derived growth factor signaling in breast cancer cells

Figure 1

The PDZ-binding motif of PTEN associates with the PDZ-I domain of NHERF1. (a) Coomassie-blue staining of glutathione S-transferase (GST)-phosphatase and tensin homolog (PTEN) fusion (arrow) and GST control protein. (b) GST pull-down assays. GST-PTEN beads were incubated with MCF7 or MDA-MB-468 cell lysates. The protein bound on beads was separated by SDS-PAGE and subjected to Na+/H+ exchanger regulatory factor 1 (NHERF1) immunoblotting. Ten per cent of the initial protein input was run side-by-side. (c) GST pull-down. The full-length NHERF1 or the PDZ-I&II domain protein was synthesized by TNT reaction in the presence of [35S]methionine. The labeled proteins were then incubated with GST or GST-PTEN. Components bound to GST fusion were separated by SDS-PAGE and detected by autoradiography. (d) Coomassie-blue staining of GST fusion of PDZ-I&II or the carboxyl-terminal half (CT) of NHERF1. (e) GST pull-down. Procedures were the same as those described for panel b except that MCF7 and T47D cells lysates, GST-PDZ-I&II and GST-CT were used in the reaction. Bound protein was detected by PTEN immunoblotting. (f) Coomassie-blue staining of GST fusion of PDZ-I or PDZ-II compared with that of PDZ-I&II. (g) NHERF1 interaction with PTEN through its PDZ-I domain. The experimental method was the same as that described for panel e except for the use of GST-PDZ-I and GST-PDZ-II. (h) PTEN binding to NHERF1 through its carboxyl-terminal PDZ-binding motif. 35S-labeled wild-type (WT) PTEN, a tumorigenic mutant (C124S), or a PTEN variant with deletion of the PDZ motif (delta C) were made by TNT reaction and evaluated for their binding to PDZ-I or PDZ-II of NHERF1. (i) Lysates from MCF7 and Zr75.1 cells were subjected to immunoprecipitation using anti-PTEN antibody or normal goat IgG. The NHERF1 protein was detected in the precipitated materials by immunoblotting. Twenty per cent of input lysates was run side-by-side.

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