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 e
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-cyclin E is a laboratory reagent used in research applications. It is an antibody that specifically binds to the cyclin E protein, which plays a key role in the regulation of the cell cycle. The core function of Anti-cyclin E is to facilitate the detection and analysis of cyclin E expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin a, by Santa Cruz Biotechnology (16 mentions)
Anti cdk4, by Santa Cruz Biotechnology (16 mentions)
Anti cdk2, by Santa Cruz Biotechnology (15 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (15 mentions)
Anti gapdh, by Santa Cruz Biotechnology (10 mentions)
Anti cyclin d, by Santa Cruz Biotechnology (9 mentions)
Anti cyclin b1, by Santa Cruz Biotechnology (8 mentions)
Anti p21, by Santa Cruz Biotechnology (8 mentions)
Anti β actin, by Santa Cruz Biotechnology (7 mentions)
Anti cyclin d1, by Cell Signaling Technology (7 mentions)
Anti p53, by Santa Cruz Biotechnology (7 mentions)
Anti akt, by Santa Cruz Biotechnology (5 mentions)
Anti cyclin b, by Santa Cruz Biotechnology (5 mentions)
Anti α tubulin, by Merck Group (4 mentions)
Anti actin, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti erk1 2, by Cell Signaling Technology (4 mentions)
Anti pcna, by Santa Cruz Biotechnology (3 mentions)
Anti p70s6k, by Santa Cruz Biotechnology (3 mentions)
Mouse and rabbit igg horseradish peroxidase conjugates, by Santa Cruz Biotechnology (3 mentions)
67 protocols using anti cyclin e
1
Western Blot Analysis of Cell Cycle Regulators
2
Mitochondrial Dynamics and Cell Cycle Analysis
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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).
3
Western Blot Analysis of Signaling Proteins
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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).
4
Western Blot Analysis of Cell Cycle Proteins
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This technique was performed as previously described11 (link). Briefly, about 40 μg of total protein from each sample were boiled for 5 min in Laemmli sample buffer and fractionated on 10 or 12% SDS–PAGE. Resolved proteins were transferred to polyvinylidene difluoride membranes and blocked for 24 h at 4 °C in blocking buffer (5% nonfat dry milk in 1% T-TBS). Then, membranes were co-incubated overnight at 4 °C with primary antibodies: anti-cyclin E (1:500, sc-377100), anti-cyclin D1 (1:500, sc-753), anti-PCNA (1:1000, sc-56), all from Santa Cruz Biotechnology (Dallas, TX, USA); anti-sGCα1 (1:1500, G4280), and anti-β-actin (1:1000, A2066) (Sigma-Aldrich, Saint Louis, MO, USA), respectively, in blocking buffer. Blots were washed and incubated for 1 h at room temperature (RT) with secondary antibodies (1:2000), used depending on the primary antibodies: goat anti-rabbit IgG-horseradish peroxidase (Jackson, West Grove, PA, USA) and goat anti-mouse IgG-horseradish peroxidase (Sigma-Aldrich). Bands were detected using ECL detection kit (Kalium, Buenos Aires, Argentina).
5
Protein Expression Analysis in Frozen Tissues
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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.
6
Western Blotting Antibody Panel
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Antibodies used for Western blotting were anti-FBW7α (#A301-720A), and anti-TRIP12 (#301-814A) from Bethyl laboratories (US), anti-vinculin (#V9131) from Sigma-Aldrich (St. Louis, MA, USA), anti-Actin HRP conjugated (#ab-49900), anti-GAPDH (#ab9485), anti-c-MYC-Y69 (#ab-32072) from Abcam (Cambridge, UK), anti-CyclinE (#sc-481) from Santa Cruz Biotechnology (Dallas, TX, USA), anti-MCL-1 (#54539) and anti-Cleaved caspase-7 (#9491) from Cell signaling technology (Denver, CO, USA).
7
Signaling Pathways in Angiogenesis
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The following agents were obtained from commercial sources: vascular endothelial growth factor‐A 165 (Merck Millipore, Billerica, MA, USA); anti‐phospho‐VEGFR‐2 (Y951) (Abcam, Cambridge, UK); anti‐phospho‐p70S6K (T421/S424), anti‐phospho‐Akt (S473), anti‐phospho‐ERK (T202/Y204), anti‐phospho‐p38MAPK (T180/Y182), anti‐phospho‐pRb (S780) and anti‐phospho‐pRb (S807/S811) (Cell Signaling, Beverly, MA, USA); anti‐phospho‐tyrosine (BD Biosciences, Bedford, MA, USA); anti‐VEGFR‐2, anti‐vascular endothelial (VE)‐cadherin, anti‐integrin β1, anti‐ILK, anti‐p70S6K, anti‐Akt, anti‐ERK, anti‐p38MAPK, anti‐Cdk2, anti‐Cdk4, anti‐cyclin D, anti‐cyclin E, anti‐actin antibodies and mouse and rabbit IgG‐horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Alexa Fluor 488–conjugated goat anti‐mouse IgG (Life Technologies, Grand Island, NY, USA).
8
PBSA Modulates Cell Signaling Pathways
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2‐Phenylbenzimidazole‐5‐sulphonic acid (PBSA) was obtained from Sigma‐Aldrich. The structure of PBSA is presented in Figure 1 A. The following chemical agents and antibodies were purchased from commercial sources: p38MAPK inhibitor, SB203580 (Cayman Chemical); anti‐phospho‐Src (Y416), anti‐Src, anti‐phospho‐ERK (T202/Y204), anti‐phospho‐Akt (S473), anti‐phospho‐p70S6K (T421/S424), anti‐phospho‐MKK3 (S189)/MKK‐6 (S207), anti‐MKK3, anti‐MKK6, anti‐phospho‐p38MAPK (T180/Y182), anti‐phospho‐pRb (S780), anti‐phospho‐pRb (S807/S811), anti‐MMP‐2 and anti‐MMP‐9 (Cell Signaling); anti‐phospho‐FAK (Y397) and anti‐FAK (BD Biosciences); anti‐ERK, anti‐Akt, anti‐p70S6K, anti‐38MAPK, anti‐TIMP‐2, anti‐Cdk4, ant‐Cdk2, anti‐cyclin D, anti‐cyclin E, anti‐integrin β1, anti‐EGFR, anti‐FGFR‐1, anti‐VEGFR‐2, anti‐actin and mouse and rabbit IgG‐horseradish peroxidase conjugates (Santa Cruz Biotechnology Inc).
9
Protein Extraction and Western Blot Analysis
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Cells and tissues were lysed by radioimmunoprecipitation (RIPA) buffer without sodium deoxycholate (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40) with various inhibitors (1 mM Na3VO4, 1 mM AEBSF, 1 mM DTT, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM NaF, 1 μg/ml pepstatin A) and cOmplete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Protein concentrations in the supernatants were detected by the DC™ protein assay kit (Bio-Rad Laboratories, Hercules, USA). The equal amounts of proteins were separated by SDS-PAGE. Proteins were detected by the following antibodies diluted by CanGetSignal Solution 1 (Takara Bio Inc.); anti-GAPDH (Bethyl Laboratories, Montgomery, TX, USA), anti-DYRK2 (Sigma-Aldrich), anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), anti-Myc, anti-Myc-Ser62 (Abcam), anti-FLAG (Sigma-Aldrich), anti-Hras, anti-cyclin E, anti-cyclin D1, and anti-cyclin D2 (Santa Cruz Biotechnology, Dallas, TX, USA). Membranes were developed by ImmunoStar LD (Fujifilm Wako Pure Chemical Co., Osaka, Tokyo) and imaged using FUSION SOLO 4 M, and analysed using Fusion Capt Advance software (M&S Instruments Inc., Osaka, Tokyo).
10
Constructing HMMR Expression and Promoter Reporters
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FLAG-tagged HMMR expression vector was constructed by inserting PCR amplified HMMR fragment into the pcDNA3 vector (Invitrogen) linked with FLAG tag at the amino terminus. The HMMR promoter luciferase reporters were made by inserting PCR-amplified HMMR promoter fragments into the pGL4-Basic vector (Promega). HepG2 and MHCC-97H liver cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Small interfering RNAs (siRNAs) were synthesized by JTS scientific or GemmaPharma. The cDNA target sequences of siRNAs and/or short hairpin RNAs (shRNAs) for HMMR and CEBPα were listed in Table S1 . Stable cell lines overexpressing HMMR shRNA were established by lentiviral transduction using pSIH-H1-Puro carrying HMMR shRNA. Anti-cyclin D1, anti-cyclin E, anti-cyclin B1 and anti-CEBPα were purchased from Santa Cruz Biotechnology; Anti-HMMR was from Proteintech.
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