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. 2010 Jun;176(6):2626–2637. doi: 10.2353/ajpath.2010.091117

Figure 6.

Figure 6

PTEN binds directly to caveolin-1. A: PTEN constructs showing: wild-type (wt) PTEN containing the 8-aa caveolin-1–binding motif; mutant PTEN construct absent the caveolin-1 binding motif (ΔPTEN 271-278); and the N terminus truncated PTEN construct absent the first 129 amino acids (ΔPTEN 1-129). B: Cell free in vitro pull-down assay. Recombinant caveolin-1-GST (rCav-1-GST) was bound to GST beads and incubated with recombinant PTEN (rPTEN-His). PTEN incubated with GST beads without caveolin-1 was used as a control. Ln 1: rCav alone; Ln 2: rPTEN alone; Ln 3: rCav-1-GST incubated with GST beads alone; Ln 4: rPTEN incubated with GST beads alone; Ln 5 rPTEN incubated with rCav-1-GST and GST beads. C: Modified pull down assay. 293 cells were transfected with a HA-tagged wild-type PTEN construct. The cells were lysed and lysates incubated with: Ln 1: wild-type PTEN-HA and GST beads; Ln 2: wtPTEN-HA, rCav-1-GST, and GST beads. Shown is Western analysis of the GST beads using a HA tag antibody. Western analysis of whole cell lysates not exposed to beads using the HA tag antibody is shown as a loading control. D: Modified pull down assay. 293 cells were transfected with either HA-tagged wtPTEN, HA-tagged mutant PTEN construct where the 8-aa residues constituting the caveolin-1 binding sequence were deleted (ΔPTEN 271-278), or empty vector. Cell lysates were incubated with rCav-1-GST and GST beads, and Western analysis of beads was performed using HA antibody and caveolin-1 antibody. Western analysis of whole cell lysates was performed as a loading control. Ln 1: empty vector; Ln 2: wtPTEN; Ln 3: ΔPTEN 271-278. E: 293 cells were transfected with either HA-tagged wtPTEN or HA-tagged mutant PTEN construct (ΔPTEN 271-278). Cell lysates were incubated with rCav-1-GST and GST beads. PTEN was immunoprecipitated using a HA-tag antibody, and Western analysis of beads was performed using HA antibody and caveolin-1 antibody. Ln 1: wtPTEN (not incubated with rCav); Ln 2: wtPTEN + rCav-1-GST; Ln 3: ΔPTEN 271-278 + rCav-1-GST. HC denotes position of heavy chain. F: PTEN-null fibroblasts were infected with an adenoviral vector containing: Ln 1: empty vector (Ad-GFP) and wild-type caveolin-1 (Ad-cav-1); Ln 2: wild-type PTEN (Ad-wtPTEN) and wild-type caveolin-1 (Ad-cav-1); Ln 3: N-terminal truncated PTEN construct (Ad-ΔPTEN 1-129) and wild-type caveolin-1. Caveolin-1 was immunoprecipitated, and Western analysis for PTEN was performed. Shown as a loading control is immunoprecipitation of caveolin-1 followed by Western blotting for caveolin-1. Data are representative of three independent experiments.