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Fig. 9. Co-transfection of COS-7 cells with GSK-3ß and MAP1B, mutated at Ser1260, Thr1265 or both, partially rescues the loss of stable microtubules. (A-F) Low power, confocal fluorescence images of COS-7 cells transfected with GSK-3ß and wild-type MAP1B and immunolabelled with antibodies against GSK-3ß (A,D), a goat polyclonal antibody that recognises all forms of MAP1B (B,E) and either pAb SuperBUGS (C) or pAb BUGS (F). Cells expressing both MAP1B and GSK-3ß are immunopositive for polyclonal antibodies SuperBUGS and BUGS. Note the localisation of GSK-3ß in the nucleus as well as the cytoplasm of transfected cells (asterisk in A and D). (G-L) Low power, confocal fluorescence images of COS-7 cells transfected with GSK-3ß and either wild-type MAP1B (G-I) or MAP1B in which Ser1260 had been replaced by valine (J-L) and immunolabelled with antibodies against: GSK-3ß (G,J), a goat polyclonal antibody that recognises all forms of MAP1B (H,K), and pAb SUP GLU, which recognises stable microtubules (I,L). In cells expressing GSK-3ß and wild-type MAP1B there is a complete loss of stable microtubules (G-I) whereas in cells expressing GSK-3ß and mutated MAP1B, in which Ser1260 has been replaced by valine, a few stable microtubules remain (arrowheads). In non-transfected cells, the numbers of stable microtubules are high (asterisks in I and L). Note the localisation of GSK-3ß in the nucleus as well as the cytoplasm of transfected cells (asterisk in G and J). (M) Histogram showing the concentration of pAb SUP GLU fluorescence in COS-7 cells transfected with wild-type MAP1B (WT), MAP1B in which Ser1260 has been mutated to valine (S1260V), MAP1B in which Thr1265 has been mutated to valine (T1265V) or MAP1B in which both Ser1260 and Thr1265 have been mutated to valine (Double). Transfection with mutated MAP1B partially rescues the loss of stable microtubules seen with wild-type MAP1B. For each bar, the values are the mean±s.e.m. of results from ten cells from three separate experiments. Bar, 10 µm.
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