Cdk6 sc 177

Manufactured by Santa Cruz Biotechnology

Sourced in United States

CDK6 (sc-177) is a lab equipment product offered by Santa Cruz Biotechnology. It is a component of the cyclin-dependent kinase (CDK) family, which are key regulators of the cell cycle. CDK6 plays a crucial role in the transition from the G1 to S phase of the cell cycle.

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4 protocols using cdk6 sc 177

Analyzing Protein Expression via Immunoblotting

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To analyze protein expression in cells, immunoblotting analysis was performed as previously described [2 (link)]. Antibodies against the following proteins were obtained from Santa Cruz Technology (California, US): Aur A (sc-25425), Aur B (sc-25426), BRCA1 (sc-6954), CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), cyclin D1 (sc-718), cyclin E (sc-247), cyclin A (SC-751). Antibodies against BRCA2 (19791-1-AP) and cyclin B1 (cs-4135) were obtained from Proteintech (Chicago, USA) and Cell Signaling Technology (Massachusetts, US), respectively. The detection of β-actin (A2228, Sigma Aldrich, St. Louis, MO) was used as a loading control. The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or of donkey anti-rabbit immunoglobulin linked to horseradish peroxidase from Cell Signaling Technology (Massachusetts, US). Immunoblotting reagents were from an electrochemiluminescence kit (Amersham Biosciences). To test whether the expression levels of proteins were regulated through proteasome-mediated degradation, cells were exposed with 20 μM MG132 (#S2619, Selleck Company, Texas, America) for 3 h and then harvested for Western blotting analysis.

Wang Y., Wang Z., Qi Z., Yin S., Zhang N., Liu Y., Liu M., Meng J., Zang R., Zhang Z, & Yang G. (2014). The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy. Molecular Cancer, 13, 94.

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Immunoblotting of Cell Cycle Regulators

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Eca 109, Eca 9706, and KYSE-510 cells were starved in serum-free medium overnight, and SHR6390 were added for 24 h. The Cell pellets and tumor tissues of xenografts were lysed using RIPA Lysis Buffer (Beyotime Biotechnology, Jiangsu, China) on ice, containing complete protease inhibitor and phosphatase inhibitor cocktail (Roche, Switzerland). Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Protein samples were diluted to equal concentrations (40 μg), and separated by electrophoresis in 10–12% SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Antibodies used were against: CDK6 (sc-177) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Rb(#9313S), pRb(#9307S), CDK4(#12790), Cyclin D1 (#2978) (Cell Signaling Technology, Boston, MA, USA); β-actin (#014M4759) (Sigma-Aldrich, USA). All antibody except CDK6 (1:100) dilutions were 1:1000. Proteins were visualized using ECL plus Western Blotting Detection Reagents (GE Healthcare). Densitometry analysis of the Western blot protein was performed using the ImageJ software.

Wang J., Li Q., Yuan J., Wang J., Chen Z., Liu Z., Li Z., Lai Y., Gao J, & Shen L. (2017). CDK4/6 inhibitor-SHR6390 exerts potent antitumor activity in esophageal squamous cell carcinoma by inhibiting phosphorylated Rb and inducing G1 cell cycle arrest. Journal of Translational Medicine, 15, 127.

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Immunoprecipitation of Cell Cycle Regulators

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HT-29 cells were lysed with NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and 0.5 mM PMSF) at 4 °C for 30 min and centrifuged. The supernatants were incubated with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology), anti-CDK2 (sc-163, Santa Cruz Biotechnology), CDK4 (sc-601, Santa Cruz Biotechnology), CDK6 (sc-177, Santa Cruz Biotechnology), cyclin D (Cat. No. 06-137, Merck KGaA, Darmstadt, Germany), or cyclin E (sc-198, Santa Cruz Biotechnology) antibodies at 4 °C overnight, and were then incubated with Protein G-conjugated FG beads^® (TAS8848N1173, Tamagawa Seiki) at 4 °C for 2 h. Beads were washed three times with NETN buffer, and immunoprecipitates were eluted with Laemmli SDS sample buffer and subjected to a western blot analysis with Clean-Blot IP Detection Reagent-HRP (Thermo Fisher Scientific) instead of secondary antibodies.

Iizumi Y., Sowa Y., Goi W., Aono Y., Watanabe M., Kurumida Y., Kameda T., Akaji K., Kitagawa M, & Sakai T. (2022). Stabilization of CDK6 by ribosomal protein uS7, a target protein of the natural product fucoxanthinol. Communications Biology, 5, 564.

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Metformin Effects on Pancreatic Cancer Cells

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Chemicals. Metformin (1,1-dimethylbiguanide) was purchased from Dainippon Sumitomo Pharma Inc. (Osaka, Japan). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan), and all other chemicals were obtained from Sigma Chemical Co. (Tokyo, Japan).
Antibodies. The antibodies used were anti-β-actin monoclonal antibody (A5441, used at 1:3,000; Sigma-Aldrich Co., St. Louis, MO, uSA), cyclin D1 (RB-9041, used at 1:1,000; Thermo Fisher Scientific K.K., Waltham, MA, USA), cyclin E (used at 1:1,000; BD Biosciences, San Jose, CA, uSA), CDK6 (sc-177, used at 1:1,000), CDK4 (sc-749, used at 1:2,500), phosphorylated retinoblastoma protein (Rb) (sc-50, used at 1:1,000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, uSA), and secondary horseradish peroxidase-linked anti-mouse and anti-rabbit IgG antibodies (used at 1:2,000; GE Healthcare, Ltd., Buckinghamshire, uK).
Cell lines and culture. The human pancreatic cancer cell lines PK1, PK9 and Panc1 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Cells were grown in RPMI-1640 medium (Gibco Invitrogen, Carlsbad, CA, uSA) supplemented with 10% fetal bovine serum (FBS) (533-69545; Wako, Japan), and penicillin-streptomycin (100 mg/l; Gibco Invitrogen) in a humidified atmosphere of 5% CO 2 at 37˚C.

Kato K., Iwama H., Yamashita T., Kobayashi K., Fujihara S., Fujimori T., Kamada H., Kobara H, & Masaki T. (2016). The anti-diabetic drug metformin inhibits pancreatic cancer cell proliferation in vitro and in vivo: Study of the microRNAs associated with the antitumor effect of metformin. Oncology reports, 35(3).

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