Total cellular protein lysates were prepared with Pierce lysis buffer (Pierce, Rockford, IL, USA) and quantified by the Bradford method. Equal amounts of total protein (20 µg) were loaded onto 10% SDS‐PAGE gels and electrophoresed at 100 V for 1 hour. Then, the separated proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA) at 80 V for 2 hours. The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. After washing with 1× TBST, the membranes were incubated with secondary anti‐mouse or anti‐rabbit IgG HRP‐linked antibodies (1:5000; Cell Signaling Technology) at room temperature for 2 hours. The blots were developed with Target LumiGLO (Cell Signaling Technology) and photographed with DNR BioImaging System (DNR, Israel).
Dna pk
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
DNA-PK is a serine/threonine protein kinase that plays a crucial role in the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair. It is composed of a catalytic subunit (DNA-PKcs) and a regulatory subunit (Ku70/Ku80 heterodimer). DNA-PK is responsible for recognizing and binding to DNA double-strand breaks, initiating the NHEJ repair process.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phospho atm, by Cell Signaling Technology (2 mentions)
P erk, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Parp1, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Mre11, by Cell Signaling Technology (2 mentions)
Rad50, by Cell Signaling Technology (2 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Ero1 lα, by Cell Signaling Technology (1 mentions)
Phospho eif2α, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
Ire1α, by Abcam (1 mentions)
Target lumiglo, by Cell Signaling Technology (1 mentions)
Grp94, by Cell Signaling Technology (1 mentions)
γh2ax, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
13 protocols using dna pk
1
Western Blot Analysis of Cellular Stress Markers
2
Protein Expression and Immunoblotting Protocol
3
Antibody Validation for Western Blot and IF
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Antibodies for Western blot and immunofluorescence staining were purchased as follows: PAK1, PAK2, PAK3, PAK4, phospho-PAK4/5/6 (S474, S602, S560), PARP1, ATM, DNA-PK, PAK5, phospho-DNA PKs (S2056), and PAK6 (all Cell Signaling Technology, Frankfurt am Main, Germany); β-actin (Sigma-Aldrich); phospho-ATM (S1981) (Rockland Immunochemicals, Gilbertsville, PA, USA); Rad51 and γH2AX (both Merck Millipore, Burlington, MA, USA), 53BP1 (Novus Biologicals, Wiesbaden Nordenstadt, Germany); goat anti-mouse AlexaFluor488 and goat anti-rabbit AlexaFluor546 (both Life Technologies, Carlsbad, CA, USA); donkey anti-rabbit HRP and sheep anti-mouse HRP (both GE Healthcare, Chicago, IL, USA).
4
Radiation-Induced DNA Damage Signaling
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Cells pretreated with nimotuzumab received irradiation as described above. Cytoplasmic and nuclear extracts were prepared according to the directions for using the nuclear and cytoplasmic extraction kit (Beyotime, Nanjing, Jiangsu, People’s Republic of China) 6 hours after irradiation. Protein concentration was determined by the BCA protein assay kit (Servicebio, Wuhan, Hubei, People’s Republic of China). Protein lysates were then separated on an 8% or 12% SDS-PAGE gel depending on the protein molecular weight and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Immunoblots were blocked in 5% protease-free bovine serum albumin for 1 hour and probed with γ-H2AX (Abcam, Cambridge, UK), EGFR and p-EGFR (Cell Signaling Technology, Beverly, MA, USA), DNA-PK (Cell Signaling Technology, Beverly, MA, USA), p-DNA-PK (Epitomics, Burlingame, CA, USA), as well as GAPDH and Lamin B1 (Goodhere, Hangzhou, Zhejiang, People’s Republic of China) antibodies. The membranes were continually incubated with appropriate horseradish peroxidase secondary antibodies (Invitrogen, Carlsbad, NM, USA) after washing, and then we detected protein with a chemiluminescence kit (Invitrogen) and visualized the bands on an X-ray film. GAPDH and Lamin B1 were used as an internal control for cytoplasmic and nuclear proteins, respectively, to balance equal loading.
5
Western Blotting Antibody Validation
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Antibodies for Western blotting were purchased as indicated: β-actin from Sigma-Aldrich (Taufkirchen, Germany) and GAPDH, DNA-PK, ATM, PARP, NBS1, ERK1/2 and phosphoERK1/2 from Cell Signaling (Frankfurt a.M., Germany). Antibody against Rad51 was from Invitrogen (Karlsruhe, Germany) and MDM2 from Santa Cruz (Heidelberg, Germany). γH2AX antibody was from Cell Signaling (Frankfurt a. M., Germany). Alexa Fluor 488 goat anti-rabbit IgG was from Invitrogen (Karlsruhe, Germany).
6
Immunoblotting Analysis of Cellular Signaling
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Protein extracts, separated by SDS-PAGE and transferred onto PVDF membranes, were probed with antibodies against LC3B, p62 (Santa Cruz Biotechnology, Dallas, TX, USA), BMI1, p38, phospho-p38, Akt, phospho-Akt, Bcl2, Mcl1, MRE11, Bax, DNA-PK, Ku70, Ku80 (Cell Signaling Technology, Beverly, MA, USA), and ATM (Gene Tex, Irvine, CA, USA). β-actin was used as an internal loading control (Santa Cruz Biotechnology). Proteins were detected using the appropriate HRP-conjugated secondary antibodies and visualized by enhanced chemiluminescence with ECL Plus reagent (Amersham Pharmacia Biotech, Arlington Heights, IL, USA).
7
Protein Extraction and Western Blot Analysis
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Total cellular proteins and nuclear proteins were extracted using lysis buffer (Pierce, Rockford, IL, USA). The method applied has been described in detail in our previous study.9 Primary antibodies, including phospho‐eIF2α, phospho‐ATM, Bcl‐2, cleaved‐caspase3, DNA‐PK, Rad50, Nbs1, Mre11, Bcl‐xL, cleaved poly(ADP‐ribose) polymerase (PARP), phospho‐p65, p65, cyclin B, phosphor‐Cdc2 (Try15), β‐actin, histone H3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), IRE‐1, ATF‐6,HIF‐1α and PERK (1:1000; Abcam), at 4°C overnight. Secondary antibodies, anti‐mouse or anti‐rabbit IgG antibody (1:1000 dilution; Cell Signaling Technology), were added and incubated at room temperature for 2 h. Target proteins on PVDF membranes were visualized with LumiGLO (Cell Signaling Technology) and captured using a DNR BioImaging System (DNR, Israel).
8
Western Blot Analysis of DNA Damage Pathway
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Western blot analysis (FUS) was performed as described in Prause et al.47 (link). DNA damage pathway was analyzed as follows. Lysates of neuronal cell cultures were prepared as described48 with modified RIPA buffer consisting of 50 mM Tris-HCl (pH 7.4), 1% Nonidet-P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaVO4, 2 mM NaF (all Sigma-Aldrich), Complete protease inhibitor cocktail (Roche). Protein amount was measured by BCA assay (Thermo Fisher Scientific). After SDS–PAGE and transfer of proteins onto nitrocellulose membranes (GE Healthcare), specific proteins were detected using the indicated primary antibodies and horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse antibodies (GE Healthcare). Detection of proteins on X-ray films (GE Healthcare) was accomplished with enhanced chemiluminescent reagent (Amersham). Antibodies were purchased as indicated: DNA-PK (#4602), PARP1 (#9542), KU80 (#2180, Cell Signaling Technology), β-actin (A5441, Sigma), phospho-DNA-PK S2056 (ab18192), phospho-DNA-PK S2056 (ab18192, Abcam), LIG1 (ab177946), KU70 (ab3114), GFP (ab290, Abcam).
9
Western Blot Analysis of DNA Damage Signaling
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Protein concentrations were determined using the BCA kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Protein lysates (20-100 μg) were electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated primary antibodies: Phospho-ATM (Ser1981) (D6H9) from rabbit (1:500, 5883, Cell Signaling, Danvers, MA), Phospho-BRCA1 (Ser1524) from rabbit (1:500, 9009, Cell Signaling), DNA-PK from rabbit (1:500, 4602, Cell Signaling), Mre11 (31H4) from rabbit (1:500, 4847, Cell Signaling) and Rad50 from rabbit (1:500, 3427, Cell Signaling). Membranes were then incubated with a 1:5,000 dilution of a peroxidase conjugated corresponding secondary antibody. Equal loading of the protein samples was confirmed by parallel western blots for β-actin (1:5,000, ab822750; Abcam). Detection was performed using the ECL-Enhanced Chemiluminescence Detection System (GE Healthcare Biosciences, Pittsburgh, PA) according to the manufacturer's instructions. Blots were visualized by autoradiography.
10
Investigating Signaling Pathways in Cancer
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Dasatinib, BIBW2992, PD0325091 and Etoposide were purchased from Selleck Chemicals (Houston, TX) and prepared as 20 mM stock solutions in DMSO. Antibodies used included anti-YAP-TAZ, pLats, Lats1, Lats2, pMST1/2, pH2AX, MEK, pMEK(S217/221), ERK, pERK(T202/Y204), pRb, pS6, LC3A/B, ATM, pChk1, Chk1, pChk2, Chk2, pCDC25c, pATRIP, p27, p21, pp38, β-catenin, cyclin D1, survivin, p4EBP1, pSMAD2(S465–467), p70S6K, pCDC2, CDC6, γH2AX, ATR, pDNA-PK(S2056) and DNA-PK, (Cell Signaling Technology, Danvers, MA); BRAF and cyclin E (Santa Cruz, Dallas, TX) and ATM, Flag M2 and β-actin (Sigma-Aldrich, St. Louis, MO); pATM(S1981) (GeneTex, Irvine, CA). Predesigned siRNAs of the target genes were purchased from Dharmacon (Pittsburgh, PA) or Thermo Scientific (Rodckford, IL). pcDNA4-Chk1-Flag and pCMV5-TOPO-3Xflag-TAZ plasmids were purchased from Addgene (Cambridge, MA). WTBRAF plasmid was provided by Dr. W. Kolch (Systems Biology Irland and The Conway Institute, University College Dublin).
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