Figure 3

Anti-GSK-3 pTyr antibodies recognise additional antigens in cell nuclei and at cell borders. (A) 22Rv1 cells cultured in normal medium until they reached 60-70% confluence were fixed and stained with anti-GSK3β (pY216) antibody (red) and anti-Ki67 (green); (B) Cells were co-transfected with a eGFP, αsh2 and βsh2 plasmids and, after 72 h, fixed and stained using anti-GSK-3β pY216 (red). Transfected cells are shown in green (indicated by arrowheads). Long exposures of the red channel (top and bottom images in middle and top right image) were taken to show that the weak cytoplasmic signal detected by anti-GSK-3β pY216 is reduced in transfected cells (best observed in the top right panel by comparing the signal intensity in the cytoplasm of transfected cells with that in nearby untransfected cells, refer to the adjacent pseudocolor scale). (C and D) Untransfected cells (C) or cells transfected with eGFP, αsh2 and βsh2 plasmids (D) were fixed and stained using a mix of anti-GSK-3α and anti-GSK-3β monoclonal antibodies (red) and anti-GSK-3α/β pY279/216 from Abcam (far red, shown as white in GFP transfected cells). Images were obtained using a confocal microscope as described in the Methods. In (C) the pY279/216 antibody staining of putative focal contacts is clearest in the lower focal plane (arrowhead), whereas total GSK-3 (red) staining is diffuse. The arrowhead in (D) indicates a transfected (GFP+) cell that does not express GSK-3 but is stained at putative focal contacts using the pY279/216 antibody. The inset shows this staining at a focal plane closer to the coverslip. In (B) and (D) the bottom right panels show DIC images of the cells. Scale Bars = 50 μm.