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).
Cdc25a
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
CDC25A is a phosphatase enzyme that plays a key role in cell cycle regulation. It catalyzes the dephosphorylation of cyclin-dependent kinases, thereby promoting cell cycle progression. This core function of CDC25A is essential for the proper coordination of cell division.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin d1, by Santa Cruz Biotechnology (5 mentions)
Cdc25b, by Santa Cruz Biotechnology (3 mentions)
Cyclin a, by Santa Cruz Biotechnology (3 mentions)
Cyclin e, by Santa Cruz Biotechnology (3 mentions)
Anti cyclin a, by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
P21cip1, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Cdc25c, by Santa Cruz Biotechnology (2 mentions)
Cyclin b1, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Erk1 2, by Santa Cruz Biotechnology (2 mentions)
Cyclin e1, by Santa Cruz Biotechnology (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Anti p62, by Proteintech (1 mentions)
Anti cyclin e, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
17 protocols using cdc25a
1
Western Blot Analysis of Cell Signaling Proteins
2
Western Blot Analysis of Cell Signaling
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Samples were lysed with EZ lysis buffer (1 M Tris at pH 7, 50% glycerol, 20% SDS, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined using the protein assay kit (Bio-Rad, 500-0006). Equal amounts of proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, 10401197). Membranes were incubated overnight at 4°C with primary antibodies and for 1 h at room temperature with secondary HRP-conjugated antibodies. The antibodies used in this study include IGF1R, BCL-2, and CDC25A from Santa Cruz Biotechnology (sc-492, sc-713, and sc-7389, respectively); p-Akt Ser473, p-IGF1R Tyr1131, and p-IGF1R Tyr1135 from Cell Signaling (4060, 3021, and 3918, respectively); and β-Actin from US Biological (A0760-40).
3
Western Blot Analysis of Cell Signaling
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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).
4
Immunoblotting and MSD Assay Protocol
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Immunodetection was performed using antibodies against pAKT (Ser473), total AKT, pRPS6 (Ser240/244), total RPS6, hexokinase II (HK2, Cell Signaling, Beverly, MA, USA), CHKA (Sigma), p21Cip1 (Cell Signaling), Cdc25A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (Chemicon, Billerica, MA, USA). Blots were revealed with peroxidase-conjugated secondary anti-rabbit antibody (GE Healthcare, Chalfont St. Giles, UK) followed by ECL chemiluminescence solution (Amersham Biosciences, St. Giles, UK). The western blot procedure used for the tumor extracts was the same used for the cell lines.
An MSD assay, more sensitive than immunoblots with tissue extracts, was used to identify target inhibition ex vivo in tissue samples. It was based on a previously described protocol with minor modifications [58 (link)]. The protein concentration was determined using a Direct Detect spectrophotometer according to the manufacturer's instructions.
An MSD assay, more sensitive than immunoblots with tissue extracts, was used to identify target inhibition ex vivo in tissue samples. It was based on a previously described protocol with minor modifications [58 (link)]. The protein concentration was determined using a Direct Detect spectrophotometer according to the manufacturer's instructions.
5
Protein-Protein Interaction Analysis via Co-IP
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Co-Immunoprecipitation (Co-IP) was performed using the Dynabeads Protein G Immunoprecipitation kit (Life Technologies, Carlsbad, CA, USA). Beads were coupled to antibodies targeting 14-3-3ε (Fisher Scientific, Waltham, MA, USA) or CDC25A (Santa Cruz Biotechnology, Dallas, TX, USA) at a ratio of 7 μg of antibody for every 1 mg of beads according to the manufacturers protocol. SCC12B.2 cells were treated with vehicle (20 mM Tris buffer, pH 7.5) or fresh peptide daily at the IC50 concentration for 48 h in Opti-MEM I Reduced Serum Medium (Invitrogen) and collected by trypsinization. One hundred mg of cells were lysed in 1× IP buffer (provided by supplier) supplemented with 100 mM sodium chloride, 2 mM dithiothreitol, 1 mM magnesium chloride and protease/phosphatase inhibitors. Immunoprecipitation reactions were carried out using 1.5 mg of antibody-coupled beads incubated with 100 mg of cell lysate at 4°C for 30 minutes. Negative controls were immunoprecipitation reactions using beads coupled to IgG isotype control antibody (rabbit), and whole lysate from immunoprecipitation input loaded directly onto a gel was used as a positive control.
6
Western Blot Analysis of Signaling Pathways
7
Cryptolepine-Mediated Topoisomerase Inhibition
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Cryptolepine with ≥98% purity was purchased from Sigma-Aldrich (St. Louis, MO, USA). Topoisomerase I and II assay kits were obtained from TopoGEN, Inc. (Buena Vista, CO, USA). Primary antibodies specific for topoisomerase I and IIα were purchased from Abcam (Cambridge, MA, USA). Phospho forms of DNA damage specific primary antibodies of ATM, ATR, BRCA1, Chk1, Chk2, γH2AX, p53 and, acetylated-p53 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for total p53, mdm2, DNA-PK, cytochrome c, Cdc25a, Cdc25b, p16, p21, CDK2, cyclin A, cyclin D1, cyclin E, vinculin, β-actin, HRP-conjugated anti-mouse, anti-rabbit, and anti-goat were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Apoptotic cells were analyzed using Annexin-V Alexa Fluor® 488 (Alexafluor488) kit from Molecular Probes (Eugene, OR, USA).
8
Western Blot Analysis of Cellular Proteins
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Cells were lysed in RIPA lysis buffer (50-mM Tris–HCl, 150-mM NaCl, 5-mM EDTA, 0.5% Nonidet P-40, and a protease and phosphatase inhibitor cocktail; Calbiochem, Darmstadt, Germany). Proteins were separated by SDS-PAGE and transferred to 0.45-μM polyvinylidene difluoride membranes (Millipore). The immunoblots were processed according to standard procedures using primary antibodies directed to EGFP, p21, GAPDH, HA, Flag, V5, HDAC3 (CST, Danvers, MA, USA), DsRed, Hsp70, p53, c-Myc, Cdc25A (Santa Cruz, Dallas, TX, USA), DDB1, Cul4A, Cul4B (Abcam, Cambridge, MA, USA), DCAF8 (Bethyl, Montgomery, TX, USA) and β-tubulin (Bioworld, Louis Park, China).
9
Maduramicin Ammonium Cytotoxicity and Apoptosis
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Maduramicin ammonium (molecular weight = 934.16, purity>97%, by HPLC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (5 mg/ml), aliquoted and stored at −80°C. Dulbecco's modified Eagle's medium (DMEM) and 0.05% trypsin-EDTA were obtained from Mediatech (Manassas, VA, USA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA). One Solution Cell Proliferation Assay Kit was from Promega (Madison, WI). Cellular DNA Flow Cytometric Analysis Kit was purchased from Roche Diagnostics (Indianapolis, IN, USA). CF488A-Annexin V and Propidium Iodide (PI) Apoptosis Assay Kit was purchased from Biotium (Hayward, CA, USA). Enhanced chemiluminescence solution was from Perkin-Elmer Life Science (Boston, MA, USA). The following antibodies were used: cyclin A, cyclin B1, cyclin D1, cyclin E, CDK1, CDK2, CDK4, CDK6, CDC25A, CDC25B, CDC25C, p21Cip1, p27Kip1, Rb, p-Rb (S807/811), survivin, Mcl-1, Bcl-2, Bcl-xL, BAX, BAK, BAD, FasL, Fas/CD95, TNFα, TNFR1, TRAIL, DR4, DR5, FLIP S/L, FADD, TRADD, RIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase 3, cleaved PARP (Cell Signaling, Beverly, MA, USA), β-tubulin (Sigma, St Louis, MO), goat anti-mouse IgG-horseradish peroxidase, and goat anti-rabbit IgG-horseradish peroxidase (Pierce, Rockford, IL, USA).
10
Molecular Mechanisms of Antioxidant Effects
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Lysates from MCF-7 cells overexpressing MnSOD and CAT or pretreated with ROS scavengers ebselen (20 μM ) or NAC (1 mM ) for 4 h were exposed to E2 (367.1pM ) for 30 min before being processed for western blotting and were probed with the following antibodies ERK1/2, p-ERK1/2, p27, p27(T157)-P, ERα, p-ERα, Jab1, TFAM, PTEN, or CDC25A (Santa Cruz, Dallas, TX, USA), anti-NRF-1 (Rockland, Limerick, PA, USA), phosphorylated AKT (p-AKT) (Ser 473) and total AKT antibodies (Cell Signaling), GAPDH, or β-actin (Sigma). For immunoprecipation experiments, total cell lysates of DMSO- or E2-treated (367.1 pM for 30 min) MCF-7 cells were immunoprecipitated (IP) with anti-NRF-1 or anti-CDC25A antibodies, and immunoblots were probed with anti-NRF-1, anti-AKT, antiphosphoserine, or antityrosine antibodies.
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