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Fig. 7. Depletion of EB1 with small interfering RNA inhibits MT regrowth from centrosomes after nocodazole washout. (A) SDS lysates from untreated HeLa cultures (1) and cultures incubated with control (2) or EB1 (3) siRNA were immunoblotted for EB1 (red), tubulin (green) and actin (green). EB1 levels were reduced in cultures treated with siRNA against EB1. Polyclonal antiserum to EB1 showed weak cross-reaction with a second slower migrating protein (red signal above actin), which was not reduced in response to EB1 siRNA. (B,C) HeLa cells incubated with siRNA against GFP as a control (B) or with siRNA against EB1 (C) were treated with nocodazole to depolymerize MTs, incubated at room temperature for the indicated times after nocodazole removal to allow regrowth of MTs and immunostained for 
-tubulin (green) and EB1 (red). Some cultures were incubated with siRNA but not treated with nocodazole (–Noc) to control for preservation of MTs during fixation and, in some cultures, the nocodazole was not washed out (–wash) to control for MT regrowth during the time that was needed for the washes (compare `–wash' to `0 minutes'). Immunofluorescence images of EB1 were taken with identical exposure times to allow comparison of fluorescence intensity between images. (D,E) Analysis of MT aster areas after 20 minutes of regrowth at room temperature. HeLa cells incubated with siRNA to GFP (a,b) or siRNA to EB1 (c,d) were immunostained for EB1 (a,c) and -tubulin (b,d). Arrows mark cells depleted of EB1; an asterisk marks an unaffected cell with regular EB1 level in the same culture. (E) The areas covered by MT asters in EB1-depleted cells were compared with the areas covered by MT asters in cells of control cultures incubated with siRNA to GFP. Control, n=37; EB1 knock down, n=15.
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