Pcdk2 t160

Manufactured by Cell Signaling Technology

Sourced in United States

PCDK2 (T160) is a laboratory reagent used to detect and quantify the phosphorylation of the threonine 160 residue in the CDK2 protein. It is a highly specific antibody that can be used in various immunoassay techniques such as Western blotting, ELISA, and immunohistochemistry.

Automatically generated - may contain errors

Lab products found in correlation

5 protocols using pcdk2 t160

Analyzing DNA Damage Response Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies against Chk1, pChk1 (S317), pChk1 (S345), pChk2 (T68), pChk2 (S516), γH2AX, pCdc2 (Y15), pCdc25c (S216), Cdc25a, phH3 (S10), PARP, cleaved PARP, 53BP1, cyclin A, cyclin B1, cyclin D, cyclinE, pCDK2 (T160) and RPA70 were purchased from Cell Signaling Technologies and pChk1 (S296) from Abcam.
Cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using a BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above

Rawlinson R, & Massey A.J. (2014). γH2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments. BMC Cancer, 14, 483.

+ Open protocol

+ Expand

Immunoblot Analysis of Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primary antibodies for immunoblot analysis that were purchased from Cell Signaling Technology include: p-RB (S807/S811) (8516S), RB (9313S) (p-Akt (S473) (4070S), Akt (4691), pS6 (S235/236) (2211), S6 (2217), cyclin E1 (4129S), p21 (2947S), p27 (2552S), CDK4 (12790), Raptor (2280S), Rictor (2140S), pCDK2 (T160) (2561S) and CDK2 (2546S). Antibodies from Santa Cruz Biotechnology include: actin (SC-47778), pERK (Y204) (SC-7383), ERK (SC-514302), cyclin D1 (SC20044), CDK2 (SC-6248) and cyclin A (SC-271682). Phospho-CDK4 (PA5-664482) antibody was purchased from Thermo Fisher. Anti-CDKN2A/p16INK4a antibody was purchased from Abcam. The whole-cell extracts were prepared by lysing the cells with RIPA lysis buffer (Santa Cruz Biotechnology, SC-24948A) in the presence of 1X Halt protease inhibitor (Thermo Fisher) and 1 mM PMSF (Sigma). The extracted proteins (20 μg) were resolved by SDS-PAGE and transferred to PVDF membranes, which were then incubated with primary antibodies at 4°C overnight, followed by incubation with HRP tagged anti-mouse or anti-rabbit secondary antibodies at room temperature up to 1 hour. An enhanced chemiluminescence kit (Thermo Fisher, 34076) was used to detect the immuno-reactive bands.

Knudsen E.S., Kumarasamy V., Ruiz A., Sivinski J., Chung S., Grant A., Vail P., Chauhan S.S., Jie T., Riall T.S, & Witkiewicz A.K. (2019). Cell cycle plasticity driven by MTOR signaling: Integral resistance to CDK4/6 inhibition in patient-derived models of pancreatic cancer. Oncogene, 38(18), 3355-3370.

+ Open protocol

+ Expand

Immunoblotting and Immunohistochemistry for Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein lysates of cells growing as a monolayer were prepared as described previously [40 (link)]. Protein concentrations were determined by the Bradford assay (Biorad). 30 μg of protein were loaded on a 4–12% Bis-Tris gel (Invitrogen) and blotted onto a nitrocellulose membrane.
Immunohistochemistry of paraffin-embedded sections was performed as described previously [45 (link)]. Antigen-retrieval consisted of microwaving in 0.01 M citrate buffer (pH 6.0) for 10 min. Immunoperoxidase-based detection was performed using the Histostain-Plus 3rd Gen IHC Detection Kit (Invitrogen/Thermo Fisher Scientific) according to manufacturer's recommendations. Cells were analyzed using an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera.
Primary antibodies used for immunoblotting and immunohistochemistry were ABL1, CDK2, pTyr (all Santa Cruz), actin (Sigma), pABL1 Y412, pAKT S473, AKT, pCDK2 T160, cleaved caspase 3, pCRKL Y207, CRKL, pKIT Y719, pMAPK p42/44 T202, pPDK1 S241, PDK1, PP2A, pS6K T389, S6K (all Cell Signaling Technologies), CIP2A, PHLPP, SET (all Bethyl Laboratories), cyclin A (Novocastra), KIT (DakoCytomation) and MAPK (Invitrogen/Thermo Fisher Scientific).

Rausch J.L., Boichuk S., Ali A.A., Patil S.S., Lee D.M., Brown M.F., Makielski K.R., Liu Y., Taguchi T., Kuan S.F, & Duensing A. (2016). Opposing roles of KIT and ABL1 in the therapeutic response of gastrointestinal stromal tumor (GIST) cells to imatinib mesylate. Oncotarget, 8(3), 4471-4483.

+ Open protocol

+ Expand

Immunoblot Analysis of Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Immunoblot Analysis of Cell Cycle Regulators

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunoblots were performed as previously described [30 (link)]. Briefly, 50 μg of protein from each sample was subjected to electrophoresis and transferred to an Immobilon P membrane (Millipore, Burlington, MA, USA). Membranes were blocked for 0.5 h in BLOTTO (5% nonfat dry milk in TBS-T; 20 mmol/L Tris, 137 mmol/L NaCl, 0.25% Tween, pH 7.6) and incubated, first, with the primary antibodies and then, with the secondary antibodies. The membranes were developed with the Renaissance chemiluminescence system (PerkinElmer Life Sciences, Inc., Boston, MA, USA). The membranes were placed in an autoradiography cassette, exposed to film and scanned. The primary antibodies used were ATR (Santa Cruz Biotechnology, Dallas, TX, USA, sc-1887), cyclin E (Santa Cruz Biotechnology, sc-240), actin (Millipore, MAB1501R), pCHK1(S345) (Cell Signaling Technology, Danvers, MA, USA, #2348), CHK1 (Santa Cruz Biotechnology, sc-7898), Wee1 (Santa Cruz Biotechnology, sc-5285), pCDK1(Y15) (BD Biosciences #612306), CDK1 (Cell Signaling Technology, #9116), pCDK2(T160) (Cell Signaling Technology, #2561), CDK2 (Santa Cruz Biotechnology, sc-6248), pH2AX(S139) (EMD Millipore, #05-636), PARP (Cell Signaling Technology, #9542), vinculin (Sigma, V9131) and actin (Sigma, St. Louis, MO, USA, Clone C4, #MAB1501R).

Chen X., Yang D., Carey J.P., Karakas C., Albarracin C., Sahin A.A., Arun B.K., Guray Durak M., Li M., Kohansal M., Bui T.N., Ha M.J., Hunt K.K, & Keyomarsi K. (2021). Targeting Replicative Stress and DNA Repair by Combining PARP and Wee1 Kinase Inhibitors Is Synergistic in Triple Negative Breast Cancers with Cyclin E or BRCA1 Alteration. Cancers, 13(7), 1656.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!