Western blotting was performed as previously described [11 (link)]. The amount of protein in the cell lines was quantified by BCA reagent (Cat.#23227, Thermo Fisher Scientific). Protein lysates were extracted from the MM cell lines after incubation with different drugs or corresponding amounts of DMSO. Proteins present in lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8–12% acrylamide gels and transferred to Immobilon®-E polyvinylidene membranes (Cat.#IEVH00005, Merck Millipore), which were performed at 100 V for 90 min. Primary antibodies used for Western Blotting were obtained from Cell Signaling Technology (Danvers, MA, USA): PI3Kα (Cat#.4249S), PI3Kβ (Cat.#3011S), PI3K γ (Cat.#5405S), panAKT (Cat.#9272), p-AKT S473 (Cat.#4058), p-AKT T308 (Cat.#2965), p-p70S6K (Cat.#9234), p-S6 (Cat.#2211), p-4E-BP1 (Cat.#2855), p-ERK (Cat.#4370), cyclin D1 (Cat.#55506), CDK4 (Cat.#12790), CDK6 (Cat.#13331), LC3B (Cat.#3868), Merck Millipore: PI3Kδ (Cat.#04-401), Santa Cruz Biotechnology (Dallas, TX, USA): cyclin A (Cat.#sc-59645), PTEN (Cat.#sc-7974), Thermo Fisher Scientific: p-PRAS40 (Cat.#44-1100G), and Sigma-Aldrich: Tubulin (Cat.#T9026), β-actin (Cat.#A5441). The primary antibodies mentioned above were diluted as per manufacturer’s instructions.
Cyclin a
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Cyclin A is a protein that plays a crucial role in the regulation of the cell cycle. It is involved in the transition from the G1 phase to the S phase and from the G2 phase to the M phase of the cell cycle. Cyclin A is an essential component of the cell division process and is widely used in various areas of biological research.
Automatically generated - may contain errors
Lab products found in correlation
Securin, by Abcam (2 mentions)
Cyclin b1, by Thermo Fisher Scientific (2 mentions)
Separase, by Abcam (2 mentions)
Cyclin e, by Thermo Fisher Scientific (2 mentions)
Aurora b, by Abcam (2 mentions)
Aurora a, by Leica camera (2 mentions)
β actin, by Abcam (2 mentions)
Tubulin, by Merck Group (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Pi3kα, by Cell Signaling Technology (1 mentions)
Pi3kβ, by Merck Group (1 mentions)
Pi3k γ, by Merck Group (1 mentions)
Pakt s473, by Merck Group (1 mentions)
P erk, by Merck Group (1 mentions)
Cyclin d1, by Merck Group (1 mentions)
Pi3kδ, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Paxillin, by Thermo Fisher Scientific (1 mentions)
Cyclin d, by Merck Group (1 mentions)
Nfkb p65, by Thermo Fisher Scientific (1 mentions)
4 protocols using cyclin a
1
Comprehensive Protein Analysis for Multiple Myeloma
2
Comprehensive Molecular Profiling of Epithelial-Mesenchymal Transition
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Cyclin F (SantaCruz, sc-515,207), cyclin A (ThermoFisher, M1-154), cyclin B (ThermoFisher, MA5-14,319), cyclin D (Sigma-Aldrich, SAB4502603), RRM2 (Abcam, ab57653), p53 (ThermoFisher, Pab 240), NFkB p65 (ThermoFisher, 33–9900), vimentin – immunofluorescence (Abcam, ab92547), vimentin – Western blot (SantaCruz, sc-373,717-AF790), N-cadherin (ThermoFisher, 3B9), RhoABC (Abcam, ab188103), ROCK-2 (Abcam, ab71598), pFAK (Sigma-Aldrich, F9176), paxillin (ThermoFisher), ZO-1 (ThermoFisher, ZO1-1A12) TSBP1 (ThermoFisher, MA5-13,398)
3
Antibody Production and Validation
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The anti-DTL antibody was produced using GST–L2DTL protein, as previously described.9 (link) The following antibodies were used: cyclin A, cyclin B1, cyclin E, CDK1, and CDK2 (Thermo Fisher Scientific); β-actin, separase, securin, and Aurora B (Abcam, Cambridge, UK); TPX2 (Sigma-Aldrich Co., St Louis, MO, USA); Aurora A (Leica, Milton Keynes, UK); and NDC80 (Proteintech, Rosemont, IL, USA).
4
Cell Cycle Protein Analysis via Western Blot
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The cells were harvested at specific time points after transient transfection, washed with phosphate-buffered saline (PBS), and subsequently lysed with lysis buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 1 µg/mL aproteinin, 1 mM phenylmethanesulfonyl fluoride). Protein samples (60 µg) were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% gels) and transferred to nitrocellulose membranes (GE Healthcare Europe GmbH, Freiburg, Germany). The membranes were then incubated with primary and secondary antibodies, and immunoreactive signals were detected using the WesternBright ECL kit (Advansta Inc., Menlo Park, CA, USA). Primary antibodies (dilution) for Western blot: cyclin A (1:100), cyclin B1 (1:100), cyclin B2 (1:200), cyclin E (1:100), CDK1 (1:100), CDK2 (1:100), and CDK4 (1:100), (Thermo Fisher Scientific); β-actin (1:10,000), separase (1:1,500), securin (1:500), and aurora B (1:500), all from Abcam (Cambridge, Milton Keynes, UK); TPX2 (Sigma-Aldrich Co.); aurora A (1:1,000; Leica, UK); and P21 (1:200; Cell Signaling Technology, Inc., Danvers, MA, USA). Secondary antibodies (dilution) for Western blot: goat anti-mouse IgG (1:10,000) and goat anti-rabbit IgG (1:10,000) (Thermo Fisher Scientific).
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