FIGURE 4.
MeCP2 is a stable component of the centrosome. A, HeLa cells were transfected with siMeCP2#1 or with the corresponding control siRNA. Four days post-transfection, centrosomes were purified, and the obtained fractions were analyzed by WB with anti-MeCP2 and anti-γ-tubulin (γ-tub.). Input (Inp.) corresponds to ∼0.6% of the whole cell extract before fractionation. Fractions 1 and 5 are the bottom and top ones, respectively (n = 3). B, Western blotting of purified centrosomes using first antibodies against MeCP2 Y120P followed by stripping of the membrane and reprobing with total anti-MeCP2. γ-tubulin was used as centrosome marker. Fractions 1 and 8 are the bottom and top rows, respectively (n = 2). C, the presence of centrosomes in fraction 6 was verified by immunostaining with γ-tubulin. D, centrosomes were purified from HeLa cells expressing exogenous GFP-MeCP2, GFP, or GFP-MeCP2-Y120D, and the obtained fractions were analyzed by WB with anti-GFP and anti-γ-tubulin. Input corresponds to 0.6% of the extract before fractionation. As above, fractions 1 and 6 are the bottom and top rows, respectively (n = 3). E and F, MRC-5 cells were treated with 10 μg/ml nocodazole for 1 h to depolymerize microtubules, fixed, and stained with antibodies against MeCP2 (E, green), Tyr(P)-120 (F, green), and γ-tubulin (red) (n = 3). Scale bars = 10 μm. The insets show the magnified centrosomes.