Recombinant Human Tubulin, Beta, GST-tagged

• Cat.No.: TUBB-1501H
• Product Overview: Recombinant full-length human TUBB1 was expressed inE. colicells using an N-terminal GST tag. MW = 76 kDa.

Specification

• Species: Human
• Source: E.coli
• Tag: GST
• Description: TUBB1 or tubulin-beta 1 protein is a major constituent of microtubules. TUBB1 interaction with microtubule-associated proteins (MAPs) such as tau is fundamental for microtubule structure and function. Previous work suggested that the "microtubule binding domain" of tau (composed of three or four imperfect 18-amino acid repeats, separated by 13- or 14-amino acid inter-repeat regions) can bind to the C-terminal ends of both alpha and beta tubulin monomers. Studies revealed that TUBB1 is the target of various antitubulin agents used in the treatment of cancer. Subsequent studies have also concluded that TUBB1 mutations in clinical samples are rare, and unlikely to contribute to drug resistance.
• Sequence: Full-length.
• Applications: Kinase Assay, Western Blot.
• Storage And Stability: Store product at -70℃. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles.

Gene Information

• Gene Name: TUBB tubulin, beta [ Homo sapiens ]
• Synonyms: TUBB; tubulin, beta; TUBB5; TUBB1; M40; MGC117247; MGC16435; OK/SW-cl.56; TUBB; beta 5-tubulin; beta-4 tubulin; beta Ib tubulin; OTTHUMP00000029069; tubulin beta-1 chain; tubulin beta-5 chain; tubulin beta polypeptide; tubulin, beta polypeptide; OTTHUMP00000029069; Tubb5; Tubulin beta-5 chain
• Gene ID: 203068
• mRNA Refseq: NM_178014
• Protein Refseq: NP_821133
• MIM: 191130
• UniProt ID: P07437
• Chromosome Location: 6p21.33
• Pathway: Gap junction; Pathogenic Escherichia coli infection – EHEC; Pathogenic Escherichia coli infection – EPEC; Cell Cycle, Mitotic
• Function: GTP binding; GTPase activity; MHC class I protein binding; nucleotide binding; structural constituent of cytoskeleton

Citation

Direct interaction of metastasis-inducing S100P protein with tubulin causes enhanced cell migration without changes in cell adhesion

Journal: Biochemical Journal    PubMed ID: 32065231    Data: 2020/3/27

Authors: Min Du, Guozheng Wang, Philip S. Rudland

Article Snippet:For Western blotting, S100P was detected using mouse anti-S100P monoclonal antibody (mAb) (1 : 50 dilution) (BD Biosciences) which showed no cross-reaction with S100A1, A2 or A4 proteins [ ].For Western blotting, S100P was detected using mouse anti-S100P monoclonal antibody (mAb) (1 : 50 dilution) (BD Biosciences) which showed no cross-reaction with S100A1, A2 or A4 proteins [ ].. Anti-tubulin (α, β), anti-actin from Sigma and anti-nonmuscle myosin IIA (NMIIA) from Covance (Princetown, NJ) were used as described previously [ , ]. α-tubulin was synthesised by recombinant means ( and ) ( ) and β-tubulin purchased from Creative BioMart (Shirley, U.S.A.).. For pulldown assays, S100P-inducible COS-7 cells were incubated with doxycycline for 16 h and lysed in 20 mM Tris–HCl pH 8.0, 150 mM NaCl and 1% (v/v) Nonidet P-40 (NP-40) with 0.5 mM CaCl 2 or 1.0 mM EGTANa 2 .For pulldown assays, S100P-inducible COS-7 cells were incubated with doxycycline for 16 h and lysed in 20 mM Tris–HCl pH 8.0, 150 mM NaCl and 1% (v/v) Nonidet P-40 (NP-40) with 0.5 mM CaCl 2 or 1.0 mM EGTANa 2 .

COS-7 S10 cells were induced for 48 h with 1 μg/ml doxycycline (S100P) or left untouched (Control) prior to seeding on ( A ) glass or ( B ) fibronectin (FN)-coated glass coverslips. After a further 48 h incubation in the same but fresh media, cells were dually immunofluorescently stained with TRITC-labelled antibodies to tubulin and FITC-labelled antibodies to End Binding Protein 3 (EB3). Nuclei were counterstained blue with DAPI. Typical images are presented. White arrows indicate the well-defined mitotic organising centres (dMTOC), red arrows indicate the clear EB3 bundles at cellular periphery. Yellow arrows indicate the MTOC that are not well-defined by EB3 staining. Bars = 50 μm. Images in B′ correspond to the enlarged regions of the highlighted cells. ( C ) The number of well-defined mitotic organising centres (dMTOC) or EB3 bundles at the cellular periphery were counted and the percentage of cells containing such structures are presented as dot plots with bars representing means. *Student's t -test, P < 0.001 when S100P expressing cells (Doxy+) were compared with controls (Doxy?). Images in C′ correspond to the quantification of how formed MTOC were quantified with cells showing (1) not visible or poorly structured organisation; (2) visible but poorly formed; (3) visable and defined; and well defined and extensive regions (Methods). Both MTOC organisation and EB3 localisation at the cell periphery were disrupted when S100P is expressed.

( A ) Western blot with anti-nonmuscle myosin IIA and anti-β-actin. There was no band at 226 kDa corresponding to nonmuscle myosin IIA (IIA) in monkey COS-7 cells compared to that seen in human HeLa and rat Rama 37 cells. The gels have been overexposed to detect even the smallest trace of NMIIA. ( B ) Three clones of S100P-inducible COS-7 cells (S7, S10, S23) were exposed to 0 (c) or 1 μg/ml (d) doxycycline for 24 h. Western blot of equal amounts of loaded proteins shows relative S100P levels. In addition to monomeric S100P at an apparent mol wt of 10 kDa (arrow), multimeric forms can also be seen in these overexposed gels. ( C ) Effects of doxycycline on the abundance of tubulin. Upper panel: Typical Western blot for β-tubulin and actin in cell lines treated without (Doxy?) or with (Doxy+) 1 μg/ml doxycycline for 24 h using both rabbit anti-β-tubulin (Abcam) and rabbit anti-actin (Abcam) (Methods). Lower panel: the relative percentage (%) ratios of band densitites of tubulin/actin in dot plots with bars representing means showed no significant difference between paired doxycycline-treated cells and control cells (Students t -test, P > 0.05). ( D ) NMIIA abundance in cell lines. Western blot was performed simultaneously using rabbit anti-nonmuscle myosin IIA (NMIIA) (Abcam), rabbit anti-β-tubulin (Abcam) and rabbit anti-actin (Abcam) to detect protein levels of NMIIA, β-tubulin and actin in cell lines as indicated from cultures treated without (?) or with (+) 1 μg/ml doxycycline for 24 h (Methods). No NMIIA was detected in the COS-7 cell lines under either condition. MCF-7 produced NMIIA, but at a lower level than MDA-MB-231 and HeLa cell lines. Extra, perhaps nonspecific protein bands were seen in HeLa cells with this anti-NMIIA antibody. ( E ) Western blot for NMIIA (IIA) and actin in S100P-inducible HeLa-A19 and COS-7 S7 S10 cells treated for 24 h with 1 μg/ml doxycycline. There was no band corresponding to NMIIA in treated or untreated COS-7 cells S10 cells. ( F ) S100P abundance in cell lines. Western blot was performed using anti-S100P antibody (R&D Systems) 1/1000 on cell lysates from cultures of Huma 62 (Human Breast benign cell line for negative control), MCF-7, COS-7 parental cells without (?) or with (+) 1 μg/ml doxycycline for 24 h and COS-7-S10 (S100P-inducible) cells with (+) doxycycline under the same conditions (Methods). S100P protein was detected only in MCF-7 and COS-7-S10 treated with doxycycline. ( G ) Cell migration assay of parental COS-7 (set at 100%) and COS-7 S7, S10 and S23 cells in the absence (Doxy?) or presence (Doxy+) of 1 μg/ml doxycycline for 24 h in dot plots with bars representing means. *Student's t -test, P < 0.01 when compared with non-induced control.

( A ) Pulldown assay. His-S100P was loaded on His-binding beads. COS-7 cell lysates were applied to control beads (His-binding beads only) (a and b) or His-S100P beads (c and d) in the presence of 1 mM CaCl 2 (a and c) or EGTA (b and d). After extensive washings, bound proteins were eluted with 300 mM imidazole from each bead and any His-S100P or α, β-tubulins were detected by Western blotting using anti-S100P (Upper panel) or anti-tubulin (Lower panel) antibodies. ( B – F ) Surface Plasmon Resonance (SPR) binding assays using α,β-tubulin immobilised on a chip and different concentrations of S100P in solution. ( B ) Typical binding curves of response units (RU) in arc seconds against time in physiological buffer of 0.15 M NaCl with 200 μM CaCl 2 and multiple cycles of different concentrations of S100P. ( C ) A typical corresponding graph of response (RU) plotted against semilog of different concentrations of S100P for calculation of dissociation constant ( K d ) at or near equilibrium for one experiment. ( D ) Typical binding curves of response (RU) in arc seconds in high salt buffer (0.5 M NaCl) with 200 μM CaCl 2 and multiple cycles of different concentrations of S100P. ( E ) Corresponding graph of response (RU) plotted against semilog of different concentrations of S100P for calculation of K d at or near equilibrium for one experiment. ( F ) Typical binding curves in physiological buffer of 0.15 M NaCl for fixed concentration of 2 μM S100P and multiple cycles of different concentrations of CaCl 2 . The values of the means ± SE from three experiments are quoted in ( G and H ). Typical binding curves of S100P (2 μM) to immobilised tubulin showing response units (RU) in arc seconds plotted against time for lower concentrations of CaCl 2 in physiological buffer containing 0.15 M NaCl. The different concentrations of CaCl 2 used were 0, 0.01, 0.03, 0.1, 0.3, 1.0, 3, 10, 30 and 100 μM. ( G ) shows that near maximal binding appeared when calcium ions reached 100 μM. ( H ) shows that no significant binding was detected when calcium ions were lower than 30 μM.