Western blot was performed as previously described (Lam et al., 2020 (link)). Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich); anti-PCNA, anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, California, United States of America); anti-PFKP, anti-LDHB, anti-Bcl-2, anti-survivin, anti-XIAP, anti-AKT, anti-CDK2, anti-CDK4, anti-CDK7, anti-Cyclin D2, H, anti-CDK4, anti-N-cadherin, anti-β-catenin (Cell Signaling Technology, Danvers, Massachusetts, United States of America); and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology)] were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a reference protein (Lam et al., 2020 (link)).
Anti cdk4
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Anti-CDK4 is a primary antibody that specifically recognizes the Cyclin-Dependent Kinase 4 (CDK4) protein. CDK4 is a serine/threonine protein kinase that plays a key role in cell cycle regulation and is involved in the G1/S transition. This antibody can be used to detect and quantify CDK4 expression in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin d1, by Cell Signaling Technology (46 mentions)
Anti cdk6, by Cell Signaling Technology (29 mentions)
Anti cdk2, by Cell Signaling Technology (21 mentions)
Anti β actin, by Cell Signaling Technology (15 mentions)
Anti bcl 2, by Cell Signaling Technology (12 mentions)
Anti akt, by Cell Signaling Technology (11 mentions)
Anti p53, by Cell Signaling Technology (9 mentions)
Anti bax, by Cell Signaling Technology (9 mentions)
Anti p27, by Cell Signaling Technology (8 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Anti gapdh, by Cell Signaling Technology (7 mentions)
Anti e cadherin, by Cell Signaling Technology (7 mentions)
Anti vimentin, by Cell Signaling Technology (7 mentions)
Anti caspase 3, by Cell Signaling Technology (7 mentions)
Anti β actin, by Santa Cruz Biotechnology (7 mentions)
Anti p21, by Cell Signaling Technology (6 mentions)
Anti p27kip1, by Cell Signaling Technology (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (6 mentions)
Anti n cadherin, by Cell Signaling Technology (5 mentions)
87 protocols using anti cdk4
1
Comprehensive Protein Expression Analysis
2
Cell Lines and Reagents for Cancer Research
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HEK 293T, HCT116, HT29, LoVo, SW480, SW620 and RKO cells were obtained from our collaborators and maintained in DMEM (GIBCO) supplemented with 10% FBS (HyClone). CSBF/C10orf99 was synthesized in Chinese Peptide Company (Hangzhou, China) with a purity > 97%. Gal1 was purchased from R&D Systems (1152-GA-050). Antibodies used in this paper were: mouse anti-myc (9E10) (# SAB4700447, Sigma-Aldrich), rabbit anti-HA (C29F4) (# 3724, Cell Signaling Technology), goat anti-Human IgG (Fc specific) (# I2136, Sigma-Aldrich), anti-cyclin D1 (# 2926, Cell Signaling Technology), anti-cyclin D3 (# 2936, Cell Signaling Technology), anti-CDK4 (# 2906, Cell Signaling Technology), anti-CDK6 (# 3136, Cell Signaling Technology), anti-phospho-Cyclin B1 (Ser147) (# 4131, Cell Signaling Technology), anti-SUSD2 rabbit polyclonal antibody (# HPA004117, Sigma-Aldrich), anti-β-tublin (# T8328, Sigma-Aldrich) and anti-GAPDH (# 2118, Cell Signaling Technology). Primary cancer tissues and paired adjacent tissues were obtained from patients under primary surgery at Peking University Cancer Hospital (Beijing, China), with patients' consent and institutional ethics approval. Fresh human tissues were fixed with 10% formalin in PBS for immunohistochemistry, or frozen in liquid nitrogen for RNA extraction. This investigation was carried out after approval by the Ethics Committee of Peking University Cancer Hospital.
3
Immunoblot Analysis of Circadian Clock and Cell Cycle Proteins
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Immunoblot analysis for U2OS cells was performed as described [85 (link)] using the following antibodies: anti-BMAL1 (14020, Cell Signaling, Danvers, MA), CRY1 (A302-614A, Bethyl Laboratories, Montgomery, TX), anti-CRY2 (13997-1-AP, Proteintech, Chicago, IL), anti-RB (9309, Cell Signaling, Danvers, MA), anti-pRB-S807/811(8516, Cell Signaling, Danvers, MA), anti-pRB-S795 (9301, Cell Signaling, Danvers, MA), anti-pRB-S780 (8180, Cell Signaling, Danvers, MA), anti-pRB-S612 (AP3236a, Abgent, San Diego, CA), anti-CCNB1 (#4138, Cell Signaling, Danvers, MA), anti-cyclin D1 (ab134175, Abcam, Cambridge, MA), anti-CDK4 (12790, Cell Signaling, Danvers, MA), anti-CDK6 (14052-1-AP, Proteintech, Chicago, IL). For liver tissue extracts, anti-CDK4 (ab137675, Abcam, Cambridge, MA), anti-RB (ab24, Abcam, Cambridge, MA), and anti-pRB-S807/811 (ABC132, MilliporeSigma, Burlington, MA) were used. Anti-GAPDH (sc25778, Santa Cruz Biotech, Dallas, TX) was used as loading control antibody for both U2OS cell and liver tissue extracts.
4
Western Blot Analysis of Protein Expression
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Anti-FLAG and anti-beta actin (Sigma-Aldrich, USA); anti-GNL1, anti-RPS20, anti-CDK1 and anti-MDM2 (Abcam, UK); anti-GFP, anti-Cyclin B1 and anti-p53 (Santa Cruz, USA); anti-p21, anti-Cyclin D1, anti-CDK4, anti-pRbSer-780, anti-Rb and anti-E2F1 (Cell Signaling Technology Inc., USA) antibodies were used in western blot analysis for checking protein expression.
5
Analyzing Cell Cycle Regulators
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Western blots were performed using Mouse anti-CDK2, anti-CDK4, anti-CDK6, and anti-cyclin D1 were purchased as part of the Cell Cycle Regulation Sampler Kit (Cell Signaling Technology, Danvers, MS, USA). Mouse anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein detection was performed with Super Signal chemiluminescence substrate (Pierce, Rockford, IL, USA).
6
Western Blot Analysis of Polyadenylation Proteins
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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).
7
Western Blot Analysis of CDK4 Protein
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Radioimmunoprecipitation assay lysis buffer was used for cell lysis and total protein extraction. The total protein content was quantified with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The protein extracts were combined with protein loading buffer and boiled for 10 min. Then, 15 μg of each protein sample was separated on a 10% sodium dodecyl sulfate polyacrylamide gel (80 V for 40 min, 120 V for 80 min) (Beyotime) and transferred to a polyvinylidene difluoride membrane (210 mA constant current, 60 min) (Millipore). The membrane was blocked with 5% skimmed milk at room temperature for 2 h and then incubated with anti-CDK4 (1:1000, Cell Signaling Technology, USA, 12790s) or anti-GAPDH (1:500, Bioss, China, bs-0755R) primary antibodies overnight at 4° C. The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase (1:5000, Bioss, bs-0295G-HRP) at room temperature for 2 h. Finally, the proteins were visualized with a Western Bright ECL Kit (Advansta, USA).
8
Comprehensive Protein Analysis Protocol
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IU1, siRNA and shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MG132 and bortezomib (Velcade) were purchased from Selleckchem (Houston, TX, USA). MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). Propidium iodide (PI) and Annexin V-FITC Apoptosis Detection Kit were purchased from Keygen Company (Nanjing, China). Dynabeads antibody coupling kit was from Life technologies. Antibodies used in this study were purchased from following sources: anti-ubiquitin (P4D1) (Santa Cruz Biotechnology); anti-PARP, anti-CDK2, anti-phospho-Rb, anti-Rb, anti-PSA, anti-Bax, anti-GFP, anti-GAPDH (Bioworld Technology, Inc., Louis Park, MN, USA); anti-CDK4, anti-CDK6, anti-phospho-MDM2, anti-P53, anti-USP14, anti-Flag, anti-cyclin D1, anti-p15, anti-p27 (Cell Signaling Technology, Beverly, MA, USA); anti-MDM2 and anti-AR (Abcam, USA).
9
Antibody Immunoblotting Panel
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10
Immunohistochemical Analysis of Cell Cycle Regulators
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Paraffin-embedded xenografts or patients’ tissue samples were incubated for 2 h at 56°C for deparaffinization. Antigens were retrieved by microwave treatment in citrate buffer for 15 min to restore antigenicity. After peroxidase activity was blocked with 3% H2O2/methanol for 10 min, sections were incubated with normal goat serum for 20 min to block non-specific antibody binding sites. Sections were incubated with the primary antibodies for 1 h at 25°C and then with biotinylated anti-rabbit/mouse IgG and peroxidase-labeled streptavidin for 10 min each. The following primary antibodies were used: Rabbit anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4 and anti-CCND1 (1:100, Cell Signaling Technology), anti-CCNE1 (1:100, R&D Systems).
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