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Figure 2 | Biology Direct

Figure 2

From: Issues associated with the use of phosphospecific antibodies to localise active and inactive pools of GSK-3 in cells

Figure 2

Anti-GSK-3α/β pS21/9 and anti-GSK-3α pS21 recognise an unidentified antigen in mitotic cells. (A and B) 22Rv1 cells cultured in normal media growing at 60-70% confluence were fixed and stained with anti-GSK3α/β (pS21/9) (red) and anti-tubulin (green) (A) or with anti-GSK-3α pS21 (red) (B); note the strong staining of mitotic cells by the pS21/9 antibodies (arrowheads). (C and D) Cells were co-transfected with eGFP, αsh2 and βsh2 plasmids and, after 72 h, fixed and stained using a mix of anti-GSK3α and anti-GSK-3β monoclonal antibodies (C and D, red) and anti-GSK-3α/β pS21/9 (C) or anti-GSK-3α pS21 (D) (both far-red, shown in white). Confocal images were obtained as described in the Methods. Arrowheads indicate mitotic transfected (GFP+) cells and asterisks indicate mitotic non-transfected cells (GFP-). In (C) and (D) the bottom right panels show DIC images of the cells. (E) Extracts from cells were cultured in medium containing 0.5% serum, normal medium (10% serum) or normal medium containing nocodazole for 18 h were probed using the indicated antibodies. Asterisks mark nonspecific antigens upregulated in nocodazole treated cells. (F) 22Rv1 cells were transfected with 100 ng TopFlash or FopFlash luciferase reporters and 15 ng pGL3 Renilla luciferase, cultured for an additional 12 h in the absence or presence of nocodazole (300 ng/ml) and then stimulated with recombinant Wnt3a (R&D Systems, 100 ng/ml) for 8 h. Firefly luciferase activity was normalised using Renilla luciferase activity. Scale bars: A, C, D = 50 μm; B = 25 μm.

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