The largest database of trusted experimental protocols

5 protocols using atm inhibitor ku55933

1

Molecular Toolbox for DNA Damage Response Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols
pWZL Neo Myr Flag Syk was purchased from addgene and subcloned into plvx3 vector. GFP-CtIP were generously provided by Dr. Junjie Chen (MD Anderson Cancer Center, TX) and subcloned into plvx3. Flag–CtIP–T847E was purchased from Addgene. Anti-FLAG agarose, 3×FLAG peptide and ATM inhibitor KU55933 were purchased from Sigma Aldrich. Olaparib was purchased from LC-lab. Syk inhibitor R406 was purchased from selleckchem.
Syk(D3Z1E, 13198) antibody, anti-SQ/TQ motif (6966) and anti-pS345 Chk1 (2348) was purchased from CST. anti-CtIP (Thr847) (p1012–847, 1:2000), and anti-CtIP (Thr327) (p1012–327, 1:2000) were purchased from PhosphoSolutions. anti-RPA32 (sc-56770) and anti-CtIP (sc-271339 for WB) were purchased from Santa Cruz; anti-FLAG (F1804) was purchased from Sigma; anti-CtIP (61141, 1:1000 for IF) was purchased from Active Motif; anti- γ-H2AX antibody(05–636) was purchase from Millipore. Anti-GAPDH (60004–1-lg) was purchased from Proteintech; anti-Rad51 (GTX100469 for IF) was purchased from Genetex. Anti-H3(17168–1, AP) was purchased from Proteintech. All the secondary antibody were purchased from Jackson ImmunoResearch.
+ Open protocol
+ Expand
2

DNA Damage Response Inhibitors Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
The following agents were used: etoposide (Sigma), aphidicolin (Sigma), ATM inhibitor KU55933 (Sigma), ATR inhibitor VE-821 (EMD Millipore), BIBR1532 ((E)-2-(3-(naphthalen-2-yl)but-2-enamido)benzoic acid, Shelleckchem) and MNNG (N-methyl N'-nitro-N-nitrosoguanidine, Toronto Research Chemicals). All agents were dissolved in DMSO and stored under sterile conditions at −20°C in dark.
+ Open protocol
+ Expand
3

Anthracycline and Inhibitor Compound Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Epirubicin, Aclarubicin, Doxorubicin, Daunorubicin, Doxorubicinone, and Dimethyl-doxorubicin were dissolved in phosphate-buffered saline (PBS) at 1 mg/ml, stored at −80°C and used at the indicated final concentrations. Anthracyclines were from various commercial sources except for Dimethyl-doxorubicin, a gift from Jacques Nefjees. Etoposide (Sigma-Aldrich) was dissolved in DMSO and used at indicated final concentrations. MG-132 (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 10 µM. ATM inhibitor KU-55933 (Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 5 µM. Human recombinant TNF (PeproTech) was dissolved in RPMI and used at a final concentration of 10 ng/ml for the indicated times. Actinomycin D (ActD, Sigma-Aldrich) was dissolved in DMSO and used at a final concentration of 5 μg/ml for the indicated times. LPS (from E. coli 055:B5, Santa Cruz) was dissolved in RPMI and used at a final concentration of 100 ng/ml. PFA (Paraformaldehyde)-fixed E. coli were prepared as before (Moura-Alves et al., 2011 (link)) in house and added to the medium at a ratio of 20 bacteria per cell. AlamarBlue cell viability assay (Invitrogen) was used to determine cell viability according to the manufacturer’s instructions.
+ Open protocol
+ Expand
4

Embryonic Fibroblast and Hepatocyte Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Mouse embryonic fibroblasts (MEFs) were isolated from 14.5-day CREB+/+ and CREBS111A embryos. Where indicated, primary MEFs were immortalized by transfection with a plasmid encoding with SV40 large T antigen (p129; a gift from Dr Janet Mertz). Primary hepatocytes were isolated from CREB+/+ and CREBS111A 12-weeks female mice using the collagenase perfusion method. Primary MEFs, immortalized MEFs, primary hepatocytes, HeLa and HEK 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forskolin (10 μM, Sigma) and IBMX (100 μM, Sigma) were treated for 90 min after serum starvation for overnight. PKA inhibitor H89 (10 μM, Sigma) and ATM inhibitor KU-55933 (10 μM, Sigma) were maintained in dimethyl sulfide (DMSO) and added to cells 30 min prior to stimulation. Calicheamicin A (CLM) was a kind gift of the Pfizer Compound Transfer Program and was maintained 4 μM stock solution in DMSO.
+ Open protocol
+ Expand
5

Reagents for Cell Culture Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols
Reagents: Reagents for cell culture were purchased from HiMedia, USA. Sodium bicarbonate, TRI reagent, Hoechst 33342, doxorubicin, ATM inhibitor (KU-55933), ATR inhibitor (NU6027), thymidine, and propidium iodide was purchased from Sigma-Aldrich, USA. Molecular biology reagents were purchased either from MBI Fermentas (USA) or from NEB (USA). Protein-G fast flow bead resin was purchased from Merck-Millipore (USA). Micro-amp Fast 96-well reaction plates (0.1 ml) and micro-amp optical adhesive films were purchased from Applied Biosystems (USA). 2X fast SYBR Green PCR master mix was purchased from Kapa Biosystems (USA). For western blotting, Immobilon-P PVDF membrane was purchased from Merck-Millipore (USA). 2 and3.
+ 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?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!