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. 2017 Oct 13;37(21):e00142-17. doi: 10.1128/MCB.00142-17

FIG 3.

FIG 3

TIE2 is associated with caveolin-1 in caveolae. (A) Description of a putative binding motif (21) of TIE2 to caveolin-1. Note that the motif is common to other RTKs. (B) Coimmunoprecipitation was performed as previously described (3), with anti-TIE2 antibody, in U251.EV, U251.Tie2, and HUVEC cultures in Ang1-containing medium. The presence of caveolin-1 in TIE2 complexes was analyzed by Western blotting. WCL, whole-cell lysates. (C) HEK293.Tie2-myc and HEK293.Cav1-GFP cells that constitutively expressed TIE2 and caveolin-1, respectively, were transfected with plCav1-GFP and plTie2-myc plasmids using the X-tremeGENE HP DNA transfection reagent (Roche Applied Science) according to the manufacturer's protocol. Forty-eight hours later, Ang1 (400 ng/ml; 30 min) was added and immunoprecipitation was performed with anti-myc tag or anti-GFP antibodies, followed by a Western blot analysis of TIE2/caveolin-1 complexes. (D) U251.Tie2-myc cells were treated with Ang1 (400 ng/ml; 30 min) or IR (10 Gy; 60 min), and immunoprecipitation was performed with anti-caveolin-1 or anti-TIE2 antibodies. Levels of TIE2, caveolin-1, and PTRF were analyzed by Western blotting. U251.EV cells were used as a control. γH2AX expression levels were used as an indicator of DNA damage after IR. (E) HEK293.Tie2-myc cells were transfected with plCav1-GFP plasmid as explained for panel C, and 48 h later, cells were transferred to chamber slides (Lab-Tek) with 60% to 70% confluence. One day after being plated, cells were serum starved for 4 h and treated with Ang1 (400 ng/ml) for 30 min. Cell fixation and immunofluorescence studies were performed as previously described (3), with anti-myc tag antibody (red fluorescence). Green fluorescence from the GFP signal was used as a reporter for caveolin-1 expression. DAPI was used for nuclear staining. Merged images are provided for visualization of TIE2/caveolin-1 nuclear complexes. Images were captured using a confocal microscope (Olympus FluoView FV1000). Bar, 10 μm. A list of antibodies and working dilutions is provided in Table S3.