Anti cdk4

Manufactured by Bioss Antibodies

Sourced in China

Anti-CDK4 is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to and detects the presence of the cyclin-dependent kinase 4 (CDK4) protein.

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5 protocols using anti cdk4

Antibody-based Protein Expression Analysis

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The following antibodies were used: anti-PLK4 (Rabbit, 1:1,000), and anti-PCNA (Mouse, 1:1,000) from Abcam (Cambridge, the United Kingdom); anti-p-p38 (Rabbit, 1:1,000), anti-p38 (Rabbit, 1:1,000), anti-p-ERK (Rabbit, 1:4,000), anti-ERK (Rabbit, 1:1,000), anti-Beclin1 (Rabbit, 1:1000), anti-Atg5 (Rabbit, 1:1000), anti-LC3B (Rabbit, 1:1000) and anti-SQSTM1/p62 (Rabbit, 1:1000) from Cell Signaling Technology (Danvers, USA); anti-caspase 3 (Rabbit, 1:500) from Proteintech (Chicago, IL, USA); anti-Bax (Rabbit, 1:1000) and anti-GAPDH (Mouse, 1:1000), anti-CDK4 (Mouse, 1:1000), anti-CDK6 (Rabbit, 1:1000) from Bioss (Beijing, China). The steps for WB were based on previously described methods17 (link).

Tian X., He Y., Qi L., Liu D., Zhou D., Liu Y., Gong W., Han Z., Xia Y., Li H., Wang J., Zhu K., Chen L., Guo H, & Zhao Q. (2023). Autophagy Inhibition Contributes to Apoptosis of PLK4 Downregulation-induced Dormant Cells in Colorectal Cancer. International Journal of Biological Sciences, 19(9), 2817-2834.

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Western Blot Analysis of Apoptosis-Related Proteins

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Cells were washed three times with cold PBS and lysed on ice with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a protease and phosphatase inhibitor cocktail. The concentrations of protein were determined using a BCA protein assay kit (Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and transferred electrophoretically onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% skimmed milk powder dissolved in TBST for 2 h at room temperature and subsequently incubated overnight at 4 °C with the appropriate primary antibodies: anti-AKT3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-cyclin D1 (1:1000, Bioss, Beijing, China), anti-CDK4 (1:1000, Bioss, Beijing, China), anti-cleaved caspase-9 (1:1000, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology) and anti-GAPDH (1:10,000, Absin, Shanghai, China). Then, the membranes were washed with TBST three times and incubated for 90 min with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. The blots were detected with an ECL chemiluminescence solution and autoradiography with X-ray film. GAPDH was used as an internal control.

Liu H.Z., Shan T.D., Han Y, & Liu X.S. (2020). Silencing long non-coding RNA CASC9 inhibits colorectal cancer cell proliferation by acting as a competing endogenous RNA of miR-576-5p to regulate AKT3. Cell Death Discovery, 6, 115.

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Immunohistochemical Analysis of Senescence and Mitophagy

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Immunohistochemistry (IHC) staining was performed to detect the expression of senescence and mitophagy proteins. After antigens repaired, all sections were incubated with the primary antibody overnight at 4°C after blockage of endogenous peroxidases. The primary antibodies were used as follows: anti-Ki67 (Maixin), anti-wtp53 (1:300, Bioss), anti-p21 (1:300, Bioss), anti-CDK2 (1:50, Abcam), anti-CDK4 (1:100, Bioss), anti-CDK6 (1:400, Bioss), anti-PHB2 (1:200, Abcam) and anti-LC3 (1:100, Abcam). Next, the sections were incubated with secondary antibody (Maixin, China) at 37°C for 30 minutes. Finally, slides were evaluated using the brown DAB precipitate (Maixin, China) and were performed with hematoxylin.
All sections were observed under the microscope (Olympus BX61, Japan) to randomly select three regions at 200× magnification. Image-Pro Plus software was used for MOD quantitative analyses (MOD = integral optical density/measurement area).

Lu Y., Li L., Chen H., Jing X., Wang M., Ge L., Yang J., Zhang M, & Tang X. (2021). Peroxiredoxin1 Knockdown Inhibits Oral Carcinogenesis via Inducing Cell Senescence Dependent on Mitophagy. OncoTargets and therapy, 14, 239-251.

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Berberine and Cisplatin Synergistic Assay

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Berberine (purity: ≥98%) and cisplatin (purity: ≥99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-MMP-2, anti-MMP-9, anti-Bcl-2, anti-Bax, anti-CyclinD1, anti-CDK4, anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-P38, and anti-phospho-P38 primary antibodies were purchased from Bioss (Beijing, China). Goat antimouse IgG and goat antirabbit IgG secondary antibodies were purchased from Life Science (Santa Cruz, CA, USA).

Gao X., Zhang C., Wang Y., Zhang P., Zhang J, & Hong T. (2021). Berberine and Cisplatin Exhibit Synergistic Anticancer Effects on Osteosarcoma MG-63 Cells by Inhibiting the MAPK Pathway. Molecules, 26(6), 1666.

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Protein Extraction and Western Blot Analysis

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Cell nuclear and cytoplasmic proteins were extracted using a nuclear-cytoplasmic protein extraction kit (#P0028, Beyotime), following the manufacturer’s instructions. Cell and tissue proteins were extracted using RIPA buffer (#P0013B, Beyotime). Protein concentrations were measured with the enhanced BCA Protein Assay Kit (#P0012, Beyotime). SDS-PAGEs of the samples (30-50 μg) were conducted with 10–15% polyacrylamide gel. The electrophoresed proteins were electro-transferred into nitrocellulose filter membranes that were blocked with Tris-buffered saline, containing 5% non-fat milk. The membranes were incubated with primary antibodies overnight at 4°C and incubated with secondary antibodies at the room temperature for 1 h. The Odyssey Infrared Imaging System (Licor Biosciences) was used to visualize the bands, and Image J software was used to analyze the relative intensities of the protein bands. The antibodies used: anti-YAP (#14074, Cell Signaling Technology), anti-pYAP (#4911, Cell Signaling Technology), anti-AMPK (#5831, Cell Signaling Technology), anti-pAMPK (#bs-4002R, Bioss), anti-CCND (#55506, Cell Signaling Technology), anti-LKB1 (#3050, Cell Signaling Technology), anti-CDK4 (#bs-0633R, Bioss), and anti-CCNB (#bs-0572R, Bioss). Antibody information is presented in Supplemental Table S2.

Qu S., Liao Q., Yu C., Chen Y., Luo H., Xia X., He D., Xu Z., Jose P.A., Li Z., Wang W.E., Lyu Q.R, & Zeng C. (2022). LKB1 suppression promotes cardiomyocyte regeneration via LKB1-AMPK-YAP axis. Bosnian Journal of Basic Medical Sciences, 22(5), 772-783.

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