Cell lysate was prepared using RIPA buffer supplemented with protease/phosphatase inhibitors. Immunoblotting experiment was conducted as described previously.37 (link) The following primary antibodies were used: p-AKT Ser473 (1:800, 4060, Cell Signaling Technology, Beverly, MA, USA), p-S6RP Ser235-/236 (1:1000, CST 4858), p-4EBP1 Thr37/46 (1:1000, CST 2855), cleaved-PARP (1:1000, CST 9546), PTEN (1:200, CST 9559), BRCA1 (1:1000, 22362-1-AP, Proteintech, Rosemont, IL, USA), RAD51 (1:500, sc-8349, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-DNA-PK (S2056, ab124918, Abcam, Cambridge, MA, USA) and Vinculin (1:10000, V9131, Sigma-Aldrich, St Louis, MO, USA).10 (link), 37 (link), 38 (link), 45 (link) Immunofluorescently labeled secondary antibodies to rabbit-IgG (Molecular Probes, Grand Island, NY, USA) or mouse-IgG (Rockland Immunochemicals, Limerick, PA, USA) was used. Western blots were imaged with LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE, USA).
P dna pk
Manufactured by Abcam
Sourced in United States
P-DNA-PK is a protein kinase that plays a central role in the repair of DNA double-strand breaks. It phosphorylates various substrates involved in the DNA damage response pathway.
Automatically generated - may contain errors
Lab products found in correlation
V9131, by Merck Group (1 mentions)
Vinculin, by Merck Group (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Sc 8349, by Santa Cruz Biotechnology (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
2 protocols using p dna pk
1
Immunoblotting Analysis of Cellular Signaling
2
Protein Expression Changes in Cell Lines and Mouse Tumors
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SW837 and CT26 cells were lysed in RIPA (radioimmunoprecipitation assay) buffer and Halt protease and phosphatase inhibitor cocktail (Fisher Scientific) 4 h, 1 day or 5 days after treatment. Mouse model tumors were harvested and dissociated 2 h after completion of treatment to assess changes in protein. Samples underwent electrophoresis and protein transfer to Polyvinylidene fluoride (PVDF) membranes and were blocked in 5% nonfat dry milk in 1X Tris buffered saline and 0.1% Tween-20 (TBST). Blots were probed with 1:1,000 dilution of antibodies against pDNA-PK (Abcam), pATM (Novus Biologicals), and Kap1 (Novus Biologicals). Protein loading was verified using 1:10,000 dilution of β-Actin (Abcam). Membranes were incubated with appropriate secondaries and imaged using enhanced chemiluminescence.
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