MK-2206 2HCl and finasteride (Fin) were purchased from Shanghai Selleck Chemicals Co., Ltd. Shanghai, China. Organic reagents such as ethanol and acetonitrile were obtained from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China. Cell Cycle Assay Kit was acquired from Dojindo, Kumamoto, Japan. Primary antibodies included anti-β-Catenin, anti-GSK3β, anti-phospho-GSK3β, anti-GAPDH, and anti-β-tubulin (Boster Biological Technology, Pleasanton, CA, United States), anti-Bcl2, anti-Bax, and anti-Cyclin D1 (Abcam plc, Cambridge, United Kingdom), anti-Akt and anti-phospho-Akt (Cell Signaling Technology, Danvers, MA, United Ststes), anti-Wnt10b, anti-CDK2, anti-CDK4, and anti-P21 (ABclonal, Wuhan, China), and anti-ki67 (Proteintech, Wuhan, China). The chemiluminescence kit was obtained from Vazyme Biotech, Nanjing, China. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection kit was acquired from Promega Corporation, Madison, WI, United States.
Anti cdk4
Manufactured by ABclonal
Sourced in China
Anti-CDK4 is a laboratory equipment product. It is a protein that plays a key role in the regulation of cell cycle progression.
Automatically generated - may contain errors
Lab products found in correlation
Anti ccnb1, by Abways (2 mentions)
Anti chek1, by Abways (2 mentions)
Anti ccne1, by Abways (2 mentions)
Anti cdk1, by Abways (2 mentions)
Chemiluminescence kit, by Vazyme (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti ki67, by Proteintech (1 mentions)
Anti gapdh, by Boster Bio (1 mentions)
Anti β tubulin, by Boster Bio (1 mentions)
Anti β catenin, by Boster Bio (1 mentions)
Cell cycle assay kit, by Dojindo Laboratories (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti cdk2, by ABclonal (1 mentions)
Anti gsk3β, by Boster Bio (1 mentions)
Anti cyclin d1, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Acetonitrile, by Sinopharm (1 mentions)
Ethanol, by Sinopharm (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti cyclin d1, by ABclonal (1 mentions)
4 protocols using anti cdk4
1
Combinatorial Therapeutic Assessment with MK-2206 and Finasteride
2
Immunoblotting Protein Expression Analysis in ESCC
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Whole cell extracts were prepared from ESCC and adjacent normal tissues and cultured cells by homogenizing cells in a lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100] containing a cocktail of protease inhibitors. The supernatant was collected after centrifugation at 12 000 g for 15 min, and subjected to Western blot as previously described [20] (link). The anti-IQGAP1 (1∶5000), anti-E-cadherin (1∶500) and anti-N-cadherin (1∶500) antibodies were purchased from BD Biosciences. The anti-cyclin D1 (1∶500), anti-cyclin B1 (1∶1000), anti-CDK1 (1∶1000) and anti-CDK4 (1∶500) antibodies were purchased from ABclonal Biotechnology. Densitometry was performed by Kodak Molecular Imaging Software.
3
Immunohistochemical Analysis of Xenograft Tissues
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Paraffin-embedded sections of mouse xenograft tissues were subjected to immunohistochemical staining. Briefly, the paraffin-embedded sections were first dewaxed and rehydrated and then subjected to antigen retrieval by high-temperature incubation. Then, the samples were incubated at 4 °C overnight with primary antibodies, including anti-CCNB1 (diluted 1:300; Abways, Shanghai, China), anti-CHEK1 (diluted 1:300; Abways), anti-CCNE1 (dilution 1:300; Abways), anti-CDK1 (dilution 1:300; Abways), and anti-CDK4 (diluted 1:300; ABclonal, Wuhan, China). Immune complexes were visualized using a MaxVision HRP‐polymer IHC Kit Detection System (MaxVision, Fuzhou, China) according to the manufacturer’s protocol. Nuclei were counterstained with hematoxylin (Beyotime Biotechnology). With an optical microscope at 200× magnification, staining was scored as reported in the literature.26 (link) Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to evaluate the staining area and density and the integrated optical density (IOD) values. The mean densitometric value in the image indicates the relative expression level of the protein.
4
Protein Expression Analysis of Cell Lines
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Two cell lines (LM3 and HepG2) in the treatment groups were treated with the IC50 concentrations at 24 hours of two drugs (CI, Cino) for 24 hours and 48 hours, respectively, then the cell lines in the control groups and the treatment groups were digested and lysed respectively. Cell lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels (NCM Biotech) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were then blocked with 5% skim milk and incubated overnight at 4 °C with the following primary detection antibodies: anti-CCNB1 (dilution 1:1000; Abways, Shanghai, China), anti-CHEK1 (dilution 1:1000; Abways), anti-CCNE1 (dilution 1:400; Abways), anti-CDK1 (dilution 1:1000; Abways), anti-CDK4 (dilution 1:1000; ABclonal, Wuhan, China) and anti-GAPDH (dilution 1:5000; ProteinTech). Next, the membranes were further incubated with species-matched secondary antibodies for one and a half hours at 37 °C and then stained with western blotting substrate (Thermo Scientific, USA). Finally, band signals were detected with an enhanced chemiluminescence detection system according to the manufacturer’s protocol (MiniChemi™ I, Sage Creation, Beijing, China).
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