Aliquots of protein extracts (20 μg) derived from THP1 monocytes, their derived macrophages, HUVECs, and HASMCs were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then immunoblotted with specific antibodies raised against the following proteins: CD68, ACAT1, ICAM1, VCAM1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABCA1, collagen 1 (Novus Biologicals, Littleton, CO, USA), collagen 3, fibronectin, arginase 1, phospho (Ser529)-NFκB, α-tubulin, MMP2 (GeneTex, Irvine, CA, USA), MMP9 (EnoGene, Atlanta, GA, USA), elastin, MARCO, selectin E (Bioss, Woburn, MA, USA), PPARγ (Signalway Antibody, College Park, MD, USA), phospho (Thr202/Tyr204)-ERK1/2, phospho (Ser/Thr)-Akt (Cell Signaling Technology, Tokyo, Japan), c-Src (Bioworld Technology, St. Louis Park, MN, USA), PI3K, Bcl2, Bax, and β-actin (Sigma). The band intensity of the immunoblot was quantified by densitometry [39 (link),40 (link),41 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link)].
α tubulin
Manufactured by GeneTex
Sourced in United States
α-tubulin is a protein that is a component of microtubules, which are cytoskeletal structures involved in various cellular processes such as cell division, intracellular transport, and cell motility. It serves as a structural element within the microtubules.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (5 mentions)
Fibronectin, by GeneTex (4 mentions)
Elastin, by Bioss Antibodies (3 mentions)
Acat1, by Santa Cruz Biotechnology (3 mentions)
Abca1, by Novus Biologicals (3 mentions)
Collagen 1, by Novus Biologicals (3 mentions)
Collagen 3, by GeneTex (3 mentions)
Chemidoc xrs imaging system, by Bio-Rad (3 mentions)
Immobilon forte western hrp substrate, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Vcam 1, by Santa Cruz Biotechnology (2 mentions)
Arginase 1, by GeneTex (2 mentions)
C src, by Bioworld Technology (2 mentions)
Icam 1, by Santa Cruz Biotechnology (2 mentions)
Lamin a c, by GeneTex (2 mentions)
γ h2ax, by GeneTex (2 mentions)
Enhanced chemiluminescence solution, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated anti rabbit or mouse igg secondary antibodies, by GeneTex (2 mentions)
Anti icam 1, by Thermo Fisher Scientific (2 mentions)
28 protocols using α tubulin
1
Protein Expression Profiling of Vascular Cells
2
Immunofluorescence Analysis of Sperm Proteins
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This study was approved by the Ethics Committee of Cathay General Hospital (IRB Approval No.: CGH-P102031). All the collected protocols have been described in our previous study. All recruited participants signed an informed consent form. Semen samples were obtained by masturbation after 3–5 days of sexual abstinence. After liquefying the semen at room temperature, routine semen analysis was performed according to the WHO 2010 criteria. After washing with 1XPBS, the sperm were spread on the slides and air-dried. The slides were treated with 0.1% Triton X-100, washed twice with Tris-buffered saline (TBS), and then incubated with SEPT14 (Proteintech, Cat No, 24590-1-AP) and α-tubulin (GeneTex, Cat No. GTX628802), and ACTN4 (GeneTex, Cat. No. GTX15648; Abcam, Cat. ab108198) antibodies for 60 min at 25 °C. After washing with TBS, the sections were incubated with secondary antibodies for 60 min at room temperature and then washed again with TBS. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. The Leica DM 2000 microscope was used for observation, and the images were acquired using the SPOT 5.0 software. All steps were performed according to our previous studies [41 (link),42 (link)].
3
Investigating KLF5 and NF-κB Signaling in Cells
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Antibodies (Abs) for KLF5, IL-6, lamin A/C, α-tubulin, and p65 were purchased from GeneTex (Irvine, California, USA). Abs for TNF-α, phospho-p65 (Ser276), phospho-p65 (Ser536), and glyceraldehyde-3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), and LPS were obtained from Sigma (St. Louis, MO, USA). A KLF5 plasmid construct was purchased from GeneTex, and a KLF5 small-interfering RNA (siRNA) construct was purchased from Ambion (Austin, TX, USA). An electrophoretic mobility shift assay (EMSA) kit for NF-κB was obtained from Roche (Indianapolis, IN, USA), and nuclear protein extraction kits were obtained from Millipore (Temecula, CA, USA).
4
Immunoblotting Analysis of Macrophage and HASMC Proteins
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Aliquots of 20 µg of protein extracts from human macrophages and HASMCs were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with the following antibodies: CD36 (R&D Systems, Minneapolis, MN), ACAT1 (Santa Cruz Biotechnology, Santa Cruz, CA), ABCA1, collagen-1 (Novus Biologicals, Littleton, CO), collagen-3, fibronectin, α-tubulin, MMP2 (GeneTex, Irvine, CA), MMP9 (EnoGene, Atlanta, GA), elastin (Bioss, Woburn, MA), or β-actin (Sigma) [16] (link)–[19] (link). The densities of the bands were measured using a Densitograph System (Ez-Capture II and CS Analyzer 3.0, ATTO, Tokyo, Japan).
5
Western Blot Analysis of Integrin Expression
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Cells were disrupted with RIPA buffer, and the total protein of the cell lysates was resolved by 12% SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane, and the membrane was incubated with antibodies. The signal of the membrane was developed with PierceTM ECL Western blotting Substrate (Thermo, Waltham, MA, USA) and analyzed with a UVP Biospectrum™ imaging system (UVP, Upland, CA, USA). The primary antibodies against integrin α2, integrin β1, GAPDH and α-tubulin were purchased from GeneTex (Irvine, CA, USA). The primary antibody against ferritin and TfR1 were purchased from Santa Cruz (Dallas, TX, USA) and Cell Signaling (Beverly, MA, USA), respectively.
6
Synthesis and Characterization of Anticancer Agents
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MPT0G211, tubastatin A (TBA), and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan), and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-Tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA). PARP and CDC2 were from Santa Cruz Biotechnology (Dallas, TX, USA).
7
Proteasome Regulation and Autophagy Modulation
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Bortezomib and carfilzomib were purchased from selleck chemicals. Other reagents were purchased from puromycin (Sigma), cycloheximide (Sigma), SUC-LLVY-AMC (Enzo Life Science), Z-Leu-Leu-Glu-AMC (Enzo Life Science), Boc-Leu-Arg-Arg-AMC (Enzo Life Science). Antibodies used were as follows: PA28α (Cell Signaling), PSMA2 (Cell Signaling), S5a (Cell signaling), PA28β (Cell Signaling), Phospho-eIF2α (Ser51) (Cell signaling), eIF2α (Cell signaling), α-tubulin (Genetex), PA28γ (Genetex), PSMB5 (Genetex), PSMB6 (Enzo life science), PSMB7 (Genetex), PSMB8 (Genetex), PSMB9 (R&D systems), PSMB10 (R&D systems), Rpt5 (Enzo life science), Ubiquitin (Cell Signaling), β-actin (Santa Cruz Technology), TCF11/NRF1 (Cell Signaling), LC3B (Cell Signaling), p62/SQSTM1 (MBL International) human Ig lambda light chain (R&D systems), actin (Sigma). pLKO.1 empty vector, shRNA vector targeting human PA28α, NRF1 siRNA (ON-TARGETplus SMARTpool), PA28α siRNA (Accell SMARTpool), and control siRNA were purchased from Dharmacon. Native Mark unstained protein standard was purchased from life technologies.
8
Protein Extraction and Western Blot Analysis
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For protein extraction, cells were washed twice with ice-cold PBS and lysed on ice in Golden lysis buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 5.95 mM EDTA, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, and 10% glycerol) supplemented with protease inhibitors (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Specific antibodies against HAS3 (SAB2101014, Sigma-Aldrich, St. Louis, MO, USA), p21 (GT8611, GeneTex, Irvine, CA, USA), α-tubulin (GT114, GeneTex, Irvine, CA, USA), ATG5 (GTX113309, GeneTex, City, CA, USA), LC3 (GTX127375, GeneTex, Irvine, CA, USA), and GAPDH (sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA) were diluted 1:2000 in Tris-buffered saline/Tween 20, and the membranes were incubated for 2 h at room temperature. Horseradish peroxidase-conjugated anti-mouse IgG (sc-2354, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) secondary antibodies were diluted 1:4000 and incubated with the membranes for 1 h at room temperature.
9
Western Blot Analysis of Protein Interactions
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Western blot was performed using previous protocols [62 (link)]. Antibodies include FLAG epitope tag (1:1000, MERCK), CYLD (1:1000, Cell Signaling), p65 (1:1000, Cell Signaling), TRAF2 (1:1000, Cell Signaling) and p84 (1:5000, GeneTex, Irvine, CA, USA), α-tubulin (1:5000, GeneTex) were used as loading control.
10
Western Blot Analysis of Orbital Tissue
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Lysates were gained from orbital tissues, which were injected with BSS (sham), hPMSCs, hPMSCsPRL-1, or steroids by homogenization with PRO-PREP solution (Intron, Gyeonggido, Korea). Equal concentration of protein were loaded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes. The membranes were incubated with anti-ICAM-1 (Thermo Fisher Scientific), TGFβ2 (GeneTex, Irvine, CA, USA), TSHR (NSJ Bioreagents, San Diego, CA, USA), or α-tubulin (GeneTex). After washing, it was incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-rabbit or mouse IgG secondary antibodies at a dilution of 1:5000 (GeneTex). The immune response bands were visualized with enhanced chemiluminescence solution (Bio-Rad Laboratories) and detected using an ImageQuant LAS 4000 (GE Healthcare Life Sciences, Little Chalfont, UK).
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