Biochemical analysis of β1-tubulin in platelets and transfected cells. (A) Immunofluorescence analysis of platelets. After being fixed in methanol and permeabilized in acetone, the peripheral blood smears were hydrated with PBS and blocked with normal goat serum. Slides were concomitantly incubated with DM1A and NB2301, and then reacted with Alexa 555-labeled anti–mouse IgG and Alexa 488-labeled anti–rabbit IgG (Invitrogen). The stained cells were examined under a BX50 fluorescence microscope with a 100×/1.35 numeric aperture oil objective (Olympus, Tokyo, Japan). Image slides were taken with a DP70 digital camera using DP Manager software (Olympus). (B) Immunoblot analysis of platelets. Whole platelet proteins were isolated using a NucleoSpin RNA/Protein kit (Macherey-Nagel, Düren, Germany), separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4% to 12% gradient acrylamide slab gels (Invitrogen), and electroblotted onto polyvinylidine difluoride membranes. The blots were incubated with RB9281 and NB2301, and reacted with horseradish peroxidase-conjugated secondary antibody. The bound antibodies were visualized using an enhanced chemiluminescent substrate. Densitometric analysis of blots performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) showed that the β1-tubulin/α-tubulin ratio was reduced by 50% in the patient compared with controls (0.75 vs 1.57 ± 0.11; n = 7). (C) Megakaryocytes derived from the peripheral blood CD34⁺ cells of a normal control and the patient were doubly stained with anti-GPIIb mouse monoclonal antibody SZ22 (not shown) and NB2301 (green). Although the distribution of β1-tubulin in the patient's megakaryocytes was normal, irregular and large bleb protrusions were observed in the patient. (D) Immunofluorescence analysis of transfected CHO cells. TUBB1-myc transfected CHO cells grown on chamber slides were doubly stained with anti-myc tag antibody and DM1A, and reacted with Alexa 488-labeled anti–rabbit IgG and Alexa 555-labeled anti–mouse IgG. Note that W318 mutants were only localized as small punctuate structures. (E) TUBB1-myc transfected CHO cells grown on chamber slides were exposed to 4°C for 4 hours to facilitate the depolymerization of microtubules, and then fixed and stained. Aggregates of β1-tubulin W318 did not dissociate under conditions of microtubule depolymerization. (F) Low temperature-exposed TUBB1-myc transfected CHO cells were lysed in 10 mM Pipes, pH 6.8, 1 mM MgCl₂, 1 mM EGTA, 10% dimethyl sulfoxide, 0.5% Nonidet P-40, and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany), and incubated for 30 minutes at 37°C. Cell lysates were centrifuged at 1000g for 10 minutes at 37°C before supernatants and pellets were collected for immunoblot analysis with anti-myc tag antibody. Densitometric analysis of the blots showed that a large proportion of β1-tubulin W318 was recovered in the pellets. The arrowhead indicates recombinant myc-tagged β1-tubulin.