Viable cells in PBS buffer or cell lysates in RIPA buffer were reduced with DTT in 2x Laemmli buffer and denatured at 100°C. Western blotting was carried out as previously described [41 (link)]. Target proteins were detected using anti-α-tubulin (1:10000, T5168, Sigma‒Aldrich, St. Louis, MO, USA), anti-TRMT112 (1:500, sc-398481, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin B1 (1:500, sc-245, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin E (1:500, sc-247, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cyclin A (1:500, sc-271682, Santa Cruz Biotechnology, Dallas, TX, USA), anti-N6AMT1 (1:1000, CQA1550, Cohesion Biosciences, London, UK), and anti-GAPDH (1:2000, sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Goat anti-rabbit-HRP (1:10000, 31460, Invitrogen, Carlsbad, CA, USA) and goat anti-mouse-HRP (1:10000, 31430, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies.
Anti cyclin a
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-cyclin A is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to the cyclin A protein, which is involved in cell cycle regulation. The core function of this product is to facilitate the detection and study of cyclin A in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin e, by Santa Cruz Biotechnology (16 mentions)
Anti cdk2, by Santa Cruz Biotechnology (8 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (8 mentions)
Anti β actin, by Santa Cruz Biotechnology (8 mentions)
Anti cdk4, by Santa Cruz Biotechnology (7 mentions)
Anti cyclin b, by Santa Cruz Biotechnology (7 mentions)
Anti gapdh, by Santa Cruz Biotechnology (6 mentions)
Anti α tubulin, by Merck Group (6 mentions)
Anti p53, by Santa Cruz Biotechnology (6 mentions)
Anti cyclin b1, by Santa Cruz Biotechnology (5 mentions)
Anti flag, by Merck Group (5 mentions)
Anti p21, by Santa Cruz Biotechnology (5 mentions)
Anti cyclin d1, by Cell Signaling Technology (4 mentions)
Anti p21, by Cell Signaling Technology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti caspase 3, by Cell Signaling Technology (4 mentions)
Anti erk, by Cell Signaling Technology (4 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (4 mentions)
Anti cdk6, by Cell Signaling Technology (4 mentions)
Anti p27, by Santa Cruz Biotechnology (4 mentions)
66 protocols using anti cyclin a
1
Western Blot Analysis of Cell Cycle Regulators
2
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein sample preparation and western blot assay were performed as previously described (24 (link)). The primary antibodies used were: anti-MTMR3 (1:500; cat. no. 12443; Cell Signaling Technology, Inc.), anti-Cyclin D1 (1:1,000; cat. no. 60186-1-1g; ProteinTech Group, Inc.), anti-cyclin-dependent kinase 2 (1:1,000; CDK2; cat. no. 11026-2-AP; ProteinTech Group, Inc.), anti-p62 (1:1,000; cat. no. 19117-1-AP; ProteinTech Group, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-Cyclin E (1:1,000; cat. no. sc-247; Santa Cruz Biotechnology, Inc.), anti-Cyclin A (1:1,000; cat. no. sc-751; Santa Cruz Biotechnology, Inc.), anti-cell division cycle 25 A (1:1,000; cdc25A; sc-7157; Santa Cruz Biotechnology, Inc.), anti-microtubule associated protein 1 light chain 3 (LC3) A (1:500; cat. no. 4599; Cell Signaling Technology, Inc.), anti-LC3B (1:500; cat. no. 3868; Cell Signaling Technology, Inc.) and anti-GAPDH (1:20,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). Bound HRP-labeled secondary antibody (1:5,000; cat. no. SC-2005 or SC-2054; Santa Cruz Biotechnology, Inc.) was assayed by super ECL detection reagent (Pierce; Thermo Fisher Scientific, Inc.). Protein density of western blots was analyzed using ImageJ 1.51k software (National Institutes of Health).
3
Mitochondrial Dynamics and Cell Cycle Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MitoTracker Green FM, DAPI, and Alexa Fluor 488– phalloidin were purchased from Life Technologies (Carlsbad, CA). Digitonin and etoposide were from Wako Pure Chemical Industries (Osaka, Japan). Mdivi-1 was from Enzo Life Sciences (Farmingdale, NY). Z-VAD-FMK was purchased from Peptide Institute (Osaka, Japan). NU6140 and BI2536 were from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were used for Western blotting and immunostaining: anti-Drp1, anti–cytochrome c (BD Biosciences, Billerica, MA), anti–γ-tubulin (Sigma-Aldrich, St. Louis, MO), anti-Smac (ProSci, Poway, CA), anti–phospho-Plk1 (Thr-210), anti-Plk1 (Abcam, Cambridge, United Kingdom), anti–phospho-CDK2 (Thr-160), anti-γ-H2AX, anti–phospho-histone H3 (Ser-10; Cell Signaling Technology, Beverly, MA), Alexa Fluor 488 anti-mouse immunoglobulin G (Life Technologies), anti–voltage-dependent anion-selective channel protein 1 (VDAC1), anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-CDK2, anti–cyclin A, anti–cyclin E, anti–cyclin B1, anti-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). The Western Lightning Plus-ECL chemiluminescence detection kit was purchased from PerkinElmer-Cetus (Boston, MA).
4
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted and equal amount of proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed overnight at 4°C with appropriate primary antibodies. Primary antibodies against the following molecules were purchased from Cell Signaling Technology (Beverly, MA, USA): EGFR, phosphorylated EGFR (Tyr992), HER2, phosphorylated HER2 (Tyr1248), HER3, phosphorylated HER3 (Tyr1289), phosphorylated STAT3 (Tyr705), phosphorylated AKT (Ser473), phosphorylated ERK (Thr202/Tyr204), PARP, caspase-3, and caspase-7. Anti-α-tubulin and anti-β-actin antibodies were purchased from Sigma-Aldrich (St. Louis). Anti-cyclin A, anti-cyclin D, and anti-cyclin E antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody binding was detected using an enhanced chemiluminescence system according to the manufacturer's protocol (Amersham Biosciences; Piscataway, NJ, USA). Anti-mouse and rabbit secondary antibodies were purchased from Thermo Scientific Inc. (Waltham, MA, USA).
5
Western Blot Analysis of Polyadenylation Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blots, protein extracts were prepared by washing and detaching ESCs as previously mentioned. Protein was extracted using RIPA buffer (50 mM Tris-HCl pH:8.8, 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% deoxycholate and 0.5% NP-40) and the concentrations quantified using the BCA Protein Assay kit (Thermo). Protein were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) (Millipore) for immunoblot detection. Primary antibodies for all the polyadenylation proteins were purchased from Bethyl Laboratories (Montgomery, TX) with the exception of CstF-64 (3A7) and τCstF-64 (6A9), which were used as described (21 (link)). Other antibodies used were rabbit polyclonal anti-FLASH (Millipore), anti-SLBP (Cell Signaling), anti-Histone H2B (Cell Signaling), anti-Cyclin A (Santa Cruz), anti-CDK2 (Cell Signaling) and anti-Histone H3 (Cell Signaling); and mouse monoclonal anti-Cyclin B1 (Millipore), anti-Histone H4 (Cell Signaling), anti-CDK4 (Cell Signaling) and anti-FLAG (Sigma).
6
Protein Expression Analysis in Frozen Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen patient tissues were homogenized in 1X RIPA buffer (Millipore, Billerica, MA, USA) supplemented with 1X Complete protease inhibitor cocktails (Roche, Mannheim, Germany). The homogenates were briefly sonicated and centrifuged at 14000rpm at 4°C for 15 minutes. The supernatant (tissue lysate) was transferred to a new vial, and 30 μg of tissue lysate from each sample was resolved by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in 1X TBST and then probed with primary antibodies at dilutions suggested by the manufacturers. Anti-DSN1, anti-SKA3, anti-UBE2C, anti-cyclin D, and anti-cyclin B1 antibodies were purchased from Abcam (Cambridge, MA, USA), anti-Aurora A was from Cell Signaling Technology (Danvers, MA, USA), anti-cyclin A and anti-cyclin E were from Santa Cruz (Santa Cruz, CA, USA), and anti-GAPDH was from Proteintech (Chicago, IL, USA). Immunoblotting images were quantified using GeneTools software (SynGene Inc., Frederick, MD, USA), and intensities of the target proteins were normalized to GAPDH.
7
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein lysates were obtained using RIPA buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 50 mM Tris (pH 7.6), and 10 μL/mL protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Equal amounts of protein from each sample were separated on SDS-PAGE gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in TBS-T (150 mM NaCl, 10 mM Tris, pH 7.4, and 0.1% Tween-20), and incubated with the following primary antibodies at 4°C overnight: poly [ADP-ribose] polymerase (PARP), cleaved PARP, CHK1, phosphorylated CHK1 (Ser345), pH2A.X, and Rad51 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-CDK 4, anti-cyclin A, -B1, -D1, -E, p21CIP1, and cdc25A (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), active caspase 3 (1:500; Epitomics, Burlingame, CA), and anti-actin (1:3000; Millipore, Billerica, MA, USA) as a loading control. After several washes with TBS-T, the membranes were incubated with HRP-conjugated secondary antibody (1:5000; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence detection system (GE Healthcare, Wauwatosa, WI, USA).
8
Immunoblot Analysis of Signaling Pathways
9
Effects of Sodium Valproate on MCF-7 Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC). Gibco media RPMI 1640 and fetal bovine serum (FBS) were used (Invitrogen Co., Carlsbad, CA, USA). Anti-caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, caspase-8, cleaved caspase-8, and p21Waf/cip1 [below as p21) antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-cyclin A, cyclin D1, cyclin E, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Cambridge, UK). Sodium valproate, Cell Counting Kit-8 (CCK-8), cell apoptosis related reagents, cell cycle detection reagents, and all other reagents used in the present study were all Sigma products (St. Louis, MO, USA).
10
Quantification of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in 35-mm coverslips and harvested at the indicated times after treatments. For 53BP1 IF, after further washing with PBS, cells were fixed with 4% PFA at RT for 10 min. Cells were subsequently permeabilized with 0.4% Triton-X100. Staining with mouse polyclonal anti-53BP1 (1:300, Millipore), γ-H2AX (1:1000 Santa Cruz Biotechnology) or rabbit polyclonal anti-Cyclin A (1:100 Santacruz), pS10H3 (1:1000 Santa Cruz Biotechnology) diluted in a 1%BSA/0,1% saponin in PBS solution, was carried out for 1 h at RT. After extensive washing with PBS, specie-specific fluorophore-conjugated antibodies (Invitrogen) were applied for 1 h at RT followed by counterstaining with 0.5 mg/ml DAPI. Secondary antibodies were used at 1:200 dilution. Images were acquired as greyscale files using Metaview software (MDS Analytical Technologies) and processed using Adobe Photoshop CS3 (Adobe). For each time point, at least 200 nuclei were examined and foci were scored at 40×.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!