The antibodies and inhibitors used are as follows: active caspase 3, poly ADP ribose and vimentin (R&D Systems); Akt, Akt-pS473, ErbB2, ErbB2-pY1248, ErK-pT202/T204, FAK-pY925 and Src-pY416 (Cell Signaling); FAK-pY397, FAK-pY576, FAK-pY577 and paxillin-pY31 (Invitrogen); E-cadherin and paxillin (BD Transduction Laboratories); anti-V5 (Serotec); FAK and N-cadherin (Santa Cruz); actin and tubulin (Sigma); FAK pY407, FAK pY861 and paxillin pY118 (Biosource); secondary antibodies (Jackson Laboratory); Hoechst 33528 (Sigma). The FAK inhibitor AZ675 was provided by AstraZeneca (Alderly Edge).
Actin and tubulin
Manufactured by Merck Group
Sourced in Canada, United States, Japan
Actin and tubulin are fundamental structural proteins found in eukaryotic cells. Actin is a globular protein that polymerizes to form microfilaments, which are essential components of the cytoskeleton. Tubulin is a heterodimeric protein that polymerizes to form microtubules, another key element of the cytoskeleton. These proteins play crucial roles in cellular processes such as cell division, intracellular transport, and cell motility.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by R&D Systems (1 mentions)
Paxillin, by BD (1 mentions)
Ps473 akt, by Cell Signaling Technology (1 mentions)
E cadherin, by BD (1 mentions)
Src py416, by Cell Signaling Technology (1 mentions)
Anti v5, by Bio-Rad (1 mentions)
Hoechst 33528, by Merck Group (1 mentions)
Erbb2, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Gel doc 1000, by Bio-Rad (1 mentions)
Anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Anti ho 1, by Stressgen (1 mentions)
Anti s18 2, by Proteintech (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Crmp4, by Abcam (1 mentions)
Oct 1, by Santa Cruz Biotechnology (1 mentions)
8 protocols using actin and tubulin
1
FAK Inhibitor Effects on Cell Signaling
2
Comprehensive Protein Expression Analysis
3
Western Blot Analysis of Nrf2 and HO-1
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Total protein extracts were prepared by treating cells with lysis buffer (RIPA). Nuclear extracts were prepared as previously described [11] (link).
Proteins were quantified using the Bradford method. Western blot analysis was performed as described previously [12] (link) using the following primary antibodies: anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1∶200 and anti-HO-1 (Stressgen, Ann Arbor, MI, USA) 1∶1000.
Nitrocellulose was then washed in TBS, and incubated with secondary antibody HRP-conjugated (dilution 1∶10000, Pierce) for 1 h, washed again and developed. The nitrocellulose was scanned using a computerized densitometric system (Bio-Rad Gel Doc 1000, Milan, Italy). Protein levels were normalized to the housekeeping protein actin and tubulin (Sigma-Aldrich) to adjust for variability of protein loading, and expressed as a percentage of vehicle control.
Proteins were quantified using the Bradford method. Western blot analysis was performed as described previously [12] (link) using the following primary antibodies: anti-Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1∶200 and anti-HO-1 (Stressgen, Ann Arbor, MI, USA) 1∶1000.
Nitrocellulose was then washed in TBS, and incubated with secondary antibody HRP-conjugated (dilution 1∶10000, Pierce) for 1 h, washed again and developed. The nitrocellulose was scanned using a computerized densitometric system (Bio-Rad Gel Doc 1000, Milan, Italy). Protein levels were normalized to the housekeeping protein actin and tubulin (Sigma-Aldrich) to adjust for variability of protein loading, and expressed as a percentage of vehicle control.
4
Western Blot Analysis of Cell Proteins
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Whole cell lysates were prepared, by boiling the equal number of cells in Laemmli buffer at 95 °C for 10 m. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer, the nitrocellulose membranes were probed overnight at 4 °C, with primary antibodies. The following primary antibodies were used: Rabbit polyclonal anti-S18-2 (Proteintech Group, Inc., Chicago, IL, USA), E-cadherin (Cell Signaling Technology, Danvers, USA), cytokeratin 8 (Dako), β-catenin (Cell signaling Technology), actin and tubulin (Sigma-Aldrich). ECL kit, anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies, produced in sheep and donkey, respectively (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), were used to visualize the protein bands.
5
Immunoblotting and Immunoprecipitation Analysis of Enteroid Extracts
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Whole cell extracts were obtained by lysing enteroids in ice-cold RIPA lysis buffer (Cell Signaling) containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, and 1% deoxycholate, supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes that were incubated with antibodies against IKKβ (Millipore, Billerica, MA), A20 (Thermofisher, Waltham, MA), IκBα, p65, (Santa Cruz Biotechnology Inc, Santa Cruz, CA), RIPK1, IKKγ, and TNF (R&D Systems, Minneapolis, MN), RIPK3 (eBioscience, Waltham, MA), cC-3, cC-8, A20, and ERK1/2 (Cell Signaling), actin, and tubulin (Sigma). For immunoprecipitation, whole cell extracts were incubated with the indicated antibodies and Protein G Dynabeads (Life Technologies) overnight at 4°C. Immunocomplexes were washed with lysis buffer and analyzed by IB. To isolate protein complexes by coimmunoprecipitation, the indicated antibodies and cell lysates were incubated in 10 mM Tris, pH 7.4, 150 mM NaCl, and 0.2% Nonident P-40.
6
Immunoprecipitation Assay for Hippocampal Neurons
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Immunoprecipitation (IP) assays were performed as described previously [24 (link)]. For hippocampal neuron immunoprecipitation, neuronal extracts were prepared by solubilization in 400 μL of cell lysis buffer for 10 min at 4°C. After a brief sonication, the lysates were centrifuged at 15,000 ×g for 10 min at 4°C. The cell extract was immunoprecipitated with 4 μg of antibodies against CRMP2 (IBL-America, Gunma, Japan), CRMP4 (Abcam), and actin and tubulin (Sigma) and incubated with 60 μL of protein G plus protein A agarose for 16 h at 4°C using continuous inversion. The immune complexes were pelleted and washed three times. The precipitated complexes were then subjected to western blot analysis.
7
Generation and Verification of p53 Mutants
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The p53 mutants S15A, S392A, 2A were generated using the QuickChange site-directed mutagenesis kit (Stratagene, USA). All of the plasmids were verified by sequencing. Antibodies were purchased from the indicated vendors: human p53 (DO-1 or Pab1801 or FL-393; Santa Cruz Biotechnology), Hdm2 (2A10; EMD Biosciences), UBE4B (BD Biosciences or RQ-5 from Santa Cruz Biotechnology), CHIP (H-231; Santa Cruz Biotechnology), Cop1 and Pirh2 (Bethyl Laboratories), phospho-p53 antibodies (#9919; Cell Signaling), Oct-1 (12F11; Santa Cruz Biotechnology), tubulin and actin (Sigma), Flag (M2 monoclonal antibody, Sigma), Myc-specific antibody (9E10; Roche), and HA (12CA5; Roche).
8
Molecular Profiling of Cellular Pathways
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Whole cell lysis, subcellular fractionation, western blot and immunoprecipitation were performed as described [17 (link), 44 (link)]. Quantification of the band density of target proteins in western blot experiments was analyzed by free software ImageJ. The following antibodies were used in western blotting and immunoprecipitation: EZH2, phospho-GSK3β Ser9, trimethyl-H3K27, and histone H3 (Cell Signaling Technology); GSK3β (BD Biosciences); dephospho-β-catenin (Ezno Life Sciences); Tubulin and Actin (Sigma); Lamin B1 (Abcam); Myc and HA (Roche). The mouse phospho-EZH2 Thr367 antibody was produced against the synthetic peptides SRPS(pT)PTINVLESKD at China Medical University Hospital in Taiwan. The synthetic peptides were obtained from LifeTein LLC. Total RNA was extracted from cells using TRIzol. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green dye on a Bio-Rad PCR machine. The primer sequences used for analysis of HOXA gene expression was listed in Supplementary Information Table S1 .
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