α tubulin

Manufactured by R&D Systems

Sourced in United States

α-tubulin is a protein that is a key component of microtubules, which are cytoskeletal structures involved in various cellular processes, such as cell division, intracellular transport, and cell motility. It is a highly conserved protein found in eukaryotic cells.

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4 protocols using α tubulin

Immunoblotting Analysis of Cell Signaling

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Cells after SC99 treatment were prepared for immunoblotting according to our previous method [35 (link), 40 (link)]. A specific primary antibody against cyclin D₂ (CCND2) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies against Bcl-2, Bcl-xL, Caspase-3, STAT3, p-STAT3(Tyr705), c-Src, p-Src, JAK2, p-JAK2, E2F-1, AKT, p-AKT, ERK, p-ERK, mTOR, p-mTOR, and PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against VEGF, β-actin, α-tubulin, anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems (Minneapolis, MN). Anti-Myc antibody and GAPDH was purchased from Sigma (St. Louis, MO).

Zhang Z., Mao H., Du X., Zhu J., Xu Y., Wang S., Xu X., Ji P., Yu Y., Cao B., Han K., Hou T., Xu Z., Kong Y., Jiang G., Tang X., Qiao C, & Mao X. (2016). A novel small molecule agent displays potent anti-myeloma activity by inhibiting the JAK2-STAT3 signaling pathway. Oncotarget, 7(8), 9296-9308.

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Protein Expression Analysis in Cells and Zebrafish

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Following the methodology published elsewhere, Western blot analysis was performed (12 (link)). Total cell lysates were extracted with RIPA buffer including a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and 20 μg of protein was utilized for Western blot analysis with VEGFA (1:1,000) and α-tubulin (1:1,000) antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Similarly, total lysates from Zebrafish embryos were extracted, and Western blot analysis was done using the antibodies Tie-2 (1:1,000) (R&D Systems, Minneapolis, MN, USA), VEGFA (1:1,000) (Abcam, Cambridge, UK), α-SMA (1:1,000) (GeneTex, Irvine, CA, USA), and α -tubulin (1:1,000) (R&D Systems). Supersignal West Dura Extended Duration Substrate (Thermo Scientific) was employed to detect the protein bands.

Vimalraj S., Subramanian R, & Dhanasekaran A. (2021). LncRNA MALAT1 Promotes Tumor Angiogenesis by Regulating MicroRNA-150-5p/VEGFA Signaling in Osteosarcoma: In-Vitro and In-Vivo Analyses. Frontiers in Oncology, 11, 742789.

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Western Blot Analysis of Protein Markers

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Our western blot procedures are described in detail elsewhere [77 (link),78 (link)]. Briefly, equal amounts of protein were separated by 10-15% SDS polyacrylamide gel electrophoresis and electro-transferred onto an Immobilon-P Transfer Membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% BSA in TBS-T and incubated with VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), leptin, α-tubulin, or Akt (R&D System), antibodies overnight at 4°C. Horseradish peroxide–conjugated secondary antibodies were hybridized by standard procedures. β-actin was used as loading control.

Hu X., Wu R., Shehadeh L.A., Zhou Q., Jiang C., Huang X., Zhang L., Gao F., Liu X., Yu H., Webster K.A, & Wang J. (2014). Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells. BMC Genomics, 15, 303.

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Protein Expression Analysis in Cell Cultures

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Cells were treated with the indicated concentrations of compounds under the indicated time. Cells were collected and lysed with RIPA buffer after being washed with cold PBS twice. The supernatant was collected after centrifugation at 16,000 × g at 4 °C for 15 min. The protein concentration was determined using BCA (Bicinchoninic Acid) assay. 20 μg total protein was loaded and then separated by SDS-PAGE. Protein was transferred to polyvinylidene fluoride (PVDF) membranes, then blocked with 5% non-fat milk, and probed by following primary antibodies: MDM2 (D1V2Z) (1:1000, CST), p53 (DO-7) (1:1000, CST), PARP (46D11) (1:1000, CST), Caspase-3 (1:1000, CST), p21 Waf1/Cip 1(12D1) (1:1000, CST), α-Tubulin(1:10000, R&D), GSPT1 (14980, 1:1000, CST), Rb (9309, 1:1000, CST), phospho-Rb (8180, 9301, CST), β-Actin (K1418, 1:1000, Santa Cruz). The membrane was washed with 1X TBS-T 3 times and incubated with HRP linked secondary antibodies for 1 h at room temperature. The membrane was incubated with ECL (Bio-rad) for 5 min. The membrane was visualized by using a Bio-Rad Chemi-Doc MP imaging system. The intensity of the band was measured by Image J software.

Wang B., Liu J., Tandon I., Wu S., Teng P., Liao J, & Tang W. (2021). Development of MDM2 Degraders Based on Ligands Derived from Ugi Reactions: Lessons and Discoveries. European journal of medicinal chemistry, 219, 113425.

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