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Blood, Vol. 113, Issue 2, 458-461, January 8, 2009
Mutation of the β1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly
Blood Kunishima et al.
113: 458
Supplemental materials for: Kunishima et al
Files in this Data Supplement:
- Table S1. Sequences and locations of oligonucleotide primers used in the PCR amplification of TUBB1 (XLS, 13.5 KB)
- Table S2. Gene frequecies of TUBB1 polymorphisms in normal controls and patients with macrothrombocytopenia (XLS, 13.5 KB)
- Table S3. Comparison of platelet size among control subjects with different TUBB1 genotypes and the patients (XLS, 13.5 KB)
- Figure S1. Characterization of NB2301 anti-human β1-tubulin antibody (JPG, 87.9 KB)
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(A) Immunoblotting of β-tubulin isoforms expressed in Escherichia coli cells. Human β-tubulin C-terminal sequences (β1-tubulin, 252451 aa; β2-tubulin, 252445 aa; β3-tubulin, 288450 aa; β4-tubulin, 333444 aa; β5-tubulin, 223444 aa; β6-tubulin, 252446 aa; β8-tubulin, 306444 aa) were cloned into a pET151/DTopo vector (Invitrogen), which has an in-frame N terminal V5-epitope-tag. Recombinant V5-tagged β-tubulins expressed in Escherichia coli BL21 (DE3) strain were analyzed by immunoblotting with anti-V5 tag antibody (Medical and Biological Laboratories, Nagoya, Japan), TUB2.1, or NB2301. (Left) The electrophoretic mobilities of V5-epitope tagged recombinant C-terminal β-tubulins were consistent with their predicted molecular weights. (Middle) TUB2.1, whose epitope is within the unspecified 281446 aa of rat brain β-tubulin, detected recombinant C-terminal β2-, β3-, β4-, β5-, and β8-tubulins. (Right) NB2301 only recognized β1-tubulin. (B) Immunofluorescence analysis. 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 NB2301 and antiβ5-tubulin antibody SAP.4G5, and then reacted with Alexa 555-labeled anti-rabbit IgG and Alexa 488-labeled anti-mouse IgG (Invitrogen). The stained cells were examined under a fluorescence microscope. NB2301 did not stain lymphocyte microtubules.
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