Fig. 3

β1tubulin is expressed at low levels in D723H cells and is incorporated in polyE-rich MTs. a Western blot of platelets and cell extracts stained for indicated antibodies. β1tubulin is expressed in mouse platelets and in D723H cells spread for 2 and 5 h on fibrinogen but is not detectable in NIH3T3-L1 nor in C2C12 mouse cell lines. Alpha tubulin and vinculin are used as a loading control. Molecular weight markers in kilodalton are indicated on the right. b D723H cell spread on fibrinogen for 16 h and stained for indicated antibodies. Representative MIP images. β1tubulin-containing MTs concentrated in the far end of the PPLLs are polyglutamylated. Bar, 30 μm. c Left panels: representative examples of β1tubulin and PolyE colocalization at different stages of MT bundling in PPLLs and in an isolated barbell-like structure analyzed by STED microscopy. A single plane is shown. Bar 5 μm. Right panels: Representative examples of coiled MTs in cleaved cytoplasts stained for indicated antibodies. Cleavage is visualized by actin staining. Bar 10 μm. d MIP mature mouse megakaryocyte extending proplatelets stained with highly diluted antibodies for β1tubulin, polyE-MT, and total tubulin. Green fire blue LUTs (GFB LUT) was applied to substracted images. Polyglutamylated and β1tubulin-containing MTs are enriched toward PPL elongation tips. MIPs are shown. Bar, 12 μm. e Blood smears from healthy or β1tubulin F260S carrying patients were stained for β1tubulin and PolyE-MTs. Low magnification (top) and high magnifications (bottom) are shown. β1tubulin and PolyE-MTs are barely detectable in platelets with β1tubulin F260S mutation. Bars 20 μm