Figure 3.

Secretion of sCADM1-Fc proteins from P3U1 cells into collagen gels. A and B: three-dimensional culture of P3U1 cells and Western blot analyses of gel lysate. As schematically presented in (A), collagen gels with a 3-mm pit were made in 12-well plates, and P3U1-sCADM1-Fc or P3U1-ΔsCADM1-Fc cells were implanted in the pit. After 2 days of culture, the gel around the pit was cut into three pieces (A, B, and C) 5 mm cubed, as illustrated in A. The gel lysates prepared from the three gel cubes were immunoblotted with anti-IgG Fc portion antibody, together with purified sCADM1-Fc proteins of indicated amounts (B). C: Immunohistochemical detection of sCADM1-Fc in three-dimensional culture collagen gels. Two-day-long three-dimensional cultures were PFA-fixed and paraffin-embedded, and sections of the collagen gels were stained with anti-IgG Fc antibody. Right three panels are high-power view images of circled areas in the left panel. Scale bar = 20 μm.