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Fig. 4. SLMAP is a component of centrosomes. (A) SLMAP localization at centrosomes is not affected by nocodazole or taxol treatment. NIH 3T3 cells were untreated (a,d,g,j) or treated with either taxol (5 µM, 4 hours; b,e,h,k) to stabilize microtubules or nocodazole (6 µg/ml, 4 hours; c,f,i,l) to depolymerize microtubules. Cells were prepared for immunofluorescence microscopy and co-stained with anti-SLMAP (a,b,c), anti-
-tubulin (d,e,f) and DAP1 (g,h,i). To show that the drugs affected cytoplasmic microtubules, cells were stained with anti-
-tubulin monoclonal antibodies (j,k,l). Bar, 25 µm. (B) Centrosomes were isolated from exponentially growing NIH 3T3 cells by fractionation on a sucrose gradient as described previously (Moudjou and Bornens, 1998). Equivalent amount (15 µg) of each protein fraction was analyzed by SDS-PAGE followed by western blotting with anti-SLMAP or anti--tubulin. Lane 1 (lysed extract), lane 2 (crude centrosomes) and lanes 3-6 represent fractions obtained from sucrose density gradients.
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