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. 2011 May 27;286(29):26071–26080. doi: 10.1074/jbc.M110.214080

FIGURE 6.

FIGURE 6.

PTPα stabilizes a secreted form of NB-3 at the cell surface. A and B, increase of cell surface sNB-3-Myc when co-expressed with VSVG-PTPα in COS1 cells. After transfection with a secreted form of NB-3 (sNB-3-Myc) alone (A, −PTPα) or with both sNB-3-Myc and VSVG-PTPα cDNAs (A, +PTPα), live COS1 cells were stained with rabbit anti-Myc antibody for cell surface NB-3-Myc protein. Images were taken using the same confocal settings. In B, COS1 cells co-transfected with both sNB-3-Myc and VSVG-PTPα were stained for cell surface sNB-3-Myc and whole cell VSVG-PTPα. Note that cells expressing high level of VSVG-PTPα also exhibit high level of cell surface sNB-3-Myc (arrowhead). Scale bars, 20 μm. C–E, analysis of steady-state cell surface level of sNB-3-Myc and VSVG-PTPα in single- and double-transfected COS1 cells. Cell surface proteins were labeled with biotin and precipitated using NeutrAvidinTM Gel. Samples were blotted with anti-Myc or anti-VSVG antibodies to assess the level of sNB-3-Myc and VSVG-PTPα proteins. For quantification in D and E, levels of cell surface proteins were normalized to corresponding input proteins. Readings for single-transfected cells were given the value of 1.0. Relative surface protein levels in double-transfected cells were calculated accordingly. Results from four independent experiments (n = 4) are presented as mean ± S.E. for each panel. *, p < 0.05; ns, not significantly different; one-sample t test. α-α-Tub, anti-α-tubulin antibody. F, co-expression with VSVG-PTPα significantly reduced the sNB-3-Myc released into the culture medium. Forty-eight hours after transfection, culture medium was collected and the free sNB-3-Myc protein level in the conditioned medium was analyzed. For quantification in G, levels of sNB-3-Myc in the conditioned medium were normalized to that in the whole cell lysates. Relative protein levels in the medium of double-transfected cells were calculated as a percentage of that of the single-transfected cells. Results from four independent experiments (n = 4) are presented as mean ± S.E. ***, p < 0.001; one-sample t test.