FIGURE 1.

Site-specific STMN serine phosphorylation contributions toward microtubule maintenance during hyperosmolarity. A, Myc-tagged STMN (STMN WT), phospho-resistant STMN (STMN AAAA), or single serine-to-alanine-substituted mutants (STMN S16A, S25A, S38A, or S63A) were transiently expressed in COS-1 cells. At 48 h post-transfection, cells were fixed and stained with anti-Myc for ectopically expressed STMN (green), anti-α-tubulin for microtubules (red), and DAPI for DNA (blue). B, COS-1 cells were transfected as in A and stimulated with sorbitol (0.5 m, 60 min) before fixation and immunofluorescence analysis. C, the proportion of control or sorbitol-treated (0.5 m, 60 min) COS-1 cells, expressing Myc-tagged STMN or STMN mutants, with normal or destabilized microtubule (MT) arrays. The total cell numbers were quantified from three independent experiments. D, the expression levels of Myc-tagged WT or mutant STMN in sorbitol-treated (OS, 0.5 m, 60 min) COS-1 cells were determined by immunoblot analysis. E, soluble and polymerized tubulin fractions were prepared from sorbitol-treated (0.5 m, 60 min) COS-1 cells expressing Myc-tagged STMN or STMN mutants and immunoblotted to assess levels of tubulin and STMN expression. The levels of polymerized tubulin as a proportion of total tubulin was calculated from two independent experiments with mean values plotted. Con, control.