ShPARG HeLa cells were plated in p100 plates with a density of 1 × 106 cells per plate and transfected with JetPEI reagent (Polyplus Transfection, Illkirch, France) with the plasmid pcDNA3-FLAG-AMPkα containing the wildtype and mutant forms of AMPk alpha 1 subunit, according to the manufacturer's guide. Twenty-four hours later, cells were starved at different times with HANK Buffer. After starvation, cells were washed 2 times with PBS1x Pefabloc 0.5 mM, resuspended in 500 μl of Lysis Buffer EBC (50 mMTris-HCl pH8, 120 mMNaCl, 0.5% NP-40, 1 mM PMSF, 1 mM Na3VO4, 10 mMNaF, Protease Inhibitor Complete Mini Roche), on ice 20 min. Centrifuge 13.000 rpm 20 min 4 °C and obtained the INPUT, the rest of supernatant was incubated with 35 μl of Protein Sepharose A (PAS) 1 hour 4 °C rocking. IP of endogenous PAR polymer performed using PAR (1:50) polyclonal antibody (4336-APC-050, TREVIGEN, My). IP was incubated at 4 °C o/n rocking and centrifuged 13.000 rpm 5 min 4 ºC. Pellets were washed 4 times with 500 μl of EBC 250-120 mMNaCl. Finally the pellet was resuspended in Loading Buffer 2x and maintained at -20 ºC.
4336 apc 050
Manufactured by Bio-Techne
Sourced in United States
4336-APC-050 is a reagent product manufactured by Bio-Techne. It is a fluorescent dye-labeled antibody specific for a biological target. The core function of this product is to serve as a detection reagent in immunoassay and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Jetpei reagent, by Polyplus Transfection (1 mentions)
Ab1790, by Abcam (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Ab16048, by Abcam (1 mentions)
Universal magnetic co ip kit, by Active Motif (1 mentions)
Pefabloc, by Roche (1 mentions)
Cisplatin, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Me 180, by American Type Culture Collection (1 mentions)
Anti gapdh antibody, by Novus Biologicals (1 mentions)
Clone 2f3, by BioLegend (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
3 protocols using 4336 apc 050
1
AMPK Alpha 1 Subunit Immunoprecipitation
2
Coimmunoprecipitation of Nuclear Proteins
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For coimmunoprecipitation, nuclear extracts were prepared using the Universal Magnetic Co-IP kit (Active Motif) in the presence of protease inhibitors (aprotinin, 10 μg/ml, Sigma; Pefabloc, 1 mM, Roche Applied Science), phosphatase inhibitor (sodium fluoride, 250 μM), and PARG inhibitor (DEA, 10 μM, Trevigen). Nuclear extracts (0.5 mg) were immunoprecipitated with NuMA antibodies or nonspecific IgG (1.5 μg) overnight at 4°C, and analyzed by immunoblotting. Similar results were obtained with two different NuMA antibodies (Calbiochem, ab-2 and Bethyl Laboratories, A301-509A). Antibodies used for immunoblot were 53BP1 (Abcam, Ab36823, 1 μg/ml), γH2AX (Ser139; Millipore, clone JBW301, 1 μg/ml), Histone H2B (Abcam, Ab1790, 0.1 μg/ml), lamin B (Abcam, Ab16048, 60 ng/ml), NuMA (B1C11, 1:2, a gift from Dr Jeffrey Nickerson, UMass, Worcester, USA), P-NuMA (S395; Cell Signaling, 3429, 1:1000), and PAR (Trevigen, 4336-APC-050, 1:1000).
3
Cervical Cancer Cell Line Protocols
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Human cervical cancer cell lines SiHa and ME180 were obtained from ATCC and maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 5% humidified CO2. PARP inhibitor Olaparib (AZD2281) was purchased from Selleckchem, Houston, USA. Cisplatin was purchased from Bio Vision, CA, USA. Anti-PARP antibody [clone 46D11, #9532 (WB 1:1000 and IF 1:800)], anti-vimentin [clone D21H3, #5741 (WB 1:1000)] and cell lysis buffer were obtained from Cell Signaling Technology, MA, USA. Anti-PAR antibody [#4336-APC-050 (WB 1:1200 and IF 1:400)] was purchased from Trevigen, MD, USA. Anti-γH2A.X (p-Ser139) antibody [clone EP854(2)Y, #ab81299 (IF 1:2000)] was purchased from Abcam, Alexa flour 488 anti-γH2A.X (phospho-S139) [clone 2F3, #613405 (IF 1:400 and FACS 1:200)] antibody was purchased from Bio Legend, San Diego, California and anti-GAPDH antibody [clone 2D4A7, #NB300–328 (WB 1:3000)] was obtained from Imgenex. XRCC4 (IF 1:200) and RPA (IF 1:400) antibodies were gifted by Dr. A.S. Balajee, Columbia University, New York, USA. XRCC1 (IF 1:500), 53BP (IF 1:500), RAD51 (IF 1:100) and Ku80 (IF 1:400) antibodies were kind gift from Dr. Sathees C. Raghavan, Indian Institute of Science, Bangalore.
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