MA5-13238

antibody from Invitrogen Antibodies

Targeting: PRKDC DNA-PKC, DNA-PKcs, DNAPK, DNAPKc, DNPK1, HYRC, HYRC1, p350, p460, XRCC7

Provider product page for MA5-13238

Western blot

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [61]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [1]
Other assay [8]

Submit

Product number: MA5-13238 - Provider product page
Provider: Invitrogen Antibodies
Product name: DNA-PK Monoclonal Antibody (18-2)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA5-13238 targets DNA-PKcs in IF, IHC (P), IP, and WB applications and shows reactivity with Human and Rat samples. The MA5-13238 immunogen is human DNA-dependent Protein Kinase purified from HeLa cells.
Reactivity: Human, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 18-2
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

PRKDC protein structure - MA5-13238 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of DNA-PKcs using DNA-PKcs Monoclonal Antibody (Product # MA5-13238) on denatured Human LS174T Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Expression of DNA-PKcs is enhanced during adipogenesis of human pre-adipocytes isolated from sub-cutaneous adipose tissue. Cells isolated from the stromal vascular fraction of collagenase digested subcutaneous tissue were grown for the indicated times in adipogenic medium as described in the Material and Methods section. (A) Photographs of the cultured cells and (B) Western blots experiments using indicated antibodies. NS, non specific band that show equal loading between the different samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1 DNA-PKcs is modified by neddylation. a DNA-PKcs was neddylated after DNA damage. Either SFB- NEDD8 WT or SFB-NEDD8DeltaG was transfected into HEK293T cells, 24 h after transfection, the cells were exposed to 10 Gy ionizing radiation. Four hours after IR treatment, cells were then harvested and subjected to Streptavidin pull-down and western blot. The blots were incubated with indicated antibodies. b MLN4924 treatment abolished the DNA-PKcs neddylation. SFB-NEDD8-transfected HEK293T cells were pretreated with 3 mu m MLN4924 for 1 h, and cells were treated with 10 Gy IR and harvested for Streptavidin pull-down and western blot. c NEDP1 is required for DNA-PKcs de-neddylation. HEK293T cells were either transfected SFB-NEDD8 solely or co-transfected with NEDP1. The cells were harvested for Streptavidin pull-down and western blot 4 h after 10 Gy IR treatment. DNA-PKcs neddylation was detected by IP-western blot. d , e Endogenous DNA-PKcs was neddylated. Endogenous DNA-PKcs or endogenous NEDD8 was immunoprecipitated with specific antibodies, respectively, and the immunoprecipitated proteins were detected by either NEDD8 antibody or DNA-PKcs antibody. f Endogenous DNA-PKcs neddylation was inhibited by MLN4924 treatment. Hela cells were pretreated with 3 mu m MLN4924 for 1 h and subjected for 10 Gy IR treatment. Six hours later the cells were harvested and processed to IP-western blotting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 HUWE1 is the DNA-PKcs neddylation E3. a , b HUWE1 interacts with DNA-PK complex and UBE2M. Immunoprecipitation was performed with DNA-PKcs antibody or Flag-HUWE1, either HUWE1 or DNA-PKcs, Ku70/Ku80, UBE2M was detected by western blotting with indicated antibodies. c HUWE1 depletion abolished DNA-PKcs neddylation. HUWE1 was depleted with specific siRNAs. The depleted cells were harvested and subjected to IP with DNA-PKcs antibody following detection with NEDD8 antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 DNA-PKcs neddylation promotes DNA-PKcs Ser2056 phosphorylation. a HUWE1 depletion diminished DNA-PKcs Ser2056 phosphorylation. HUWE1 shRNA-depleted cells were treated with VP-16 and harvested at indicated timepoints, then DNA-PKcs Ser2056 phosphorylation or ATM Ser1981 phosphorylation was examined in western blotting. b UBA3 knockdown abolished DNA-PKcs Ser2056 phosphorylation. UBA3 siRNA-depleted HeLa cells were treated with VP-16 and harvested at indicated timepoints, then DNA-PKcs Ser2056 phosphorylation was examined in western blotting. c , d MLN4924 treatment diminished DNA-PKcs Ser2056 phosphorylation after VP-16 or IR treatment. HeLa cells were pretreated with 3 mu m MLN4924 for 1 h and subjected to VP-16 treatment or different doses of IR treatment, then the cells were harvested followed by western blotting with DNA-PKcs Ser2056 antibody or indicated antibodies.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 XRCC4 is required for the recruitment of RIG-I to DSB sites. a HEK293T cells were transfected with empty vector or Flag-RIG-I. The cells were then lysed and immunoprecipitated with anti-Flag agarose beads. The beads were boiled and probed with indicated antibodies. b A549 cells were treated with IR (10 Gy, 1-2 h). Cells were lysed, and nuclear fractions were immunoprecipitated with anti-RIG-I antibody. The beads were treated with RNase A, boiled and blotted with indicated antibodies. c Control and XRCC4 knockdown ER-AsiSI U2OS cells were transfected with Flag-RIG-I, and then treated with 4-OHT to induce DSBs. Flag-RIG-I accumulation at DNA damage sites generated by AsiSI was detected by ChIP-qPCR. Data are presented as mean values +- SEM from three independent experiments. P values are determined by unpaired two-sided t -test. d Control and XRCC4 knockdown HEK293T cells overexpressing RIG-I were transfected with NHEJ reporter, and then cells were harvested for the NHEJ assay. Data are presented as mean values +- SEM from three independent experiments. P values are determined by unpaired two-sided t -test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. DNA-PK is modified by PAR in response to DNA damage. (A) IP with an anti-DNA-PKcs antibody was performed to detect the PAR modification of DNA-PKcs in HeLa cells after treatment with different irradiation doses (0, 0.5, 1, 2, 4, 6 and 8 Gy). The cells were harvested 2 h after irradiation and then lysed. (B) Western blotting was used to detect DNA-PKcs in the IP product of the anti-PAR antibody from HeLa cells treated with or without irradiation. (C) The effect of olaparib on the PARylation of DNA-PKcs. HeLa cells were treated with different concentrations of olaparib. After 24 h, the cells were harvested and lysed, and IP was used to detected the PAR modification of DNA-PKcs. (D) The effect of olaparib on the chromatin binding of DNA-PKcs in irradiated HeLa cells. DNA-PKcs, DNA-dependent protein kinase catalytic subunit; PAR, poly(ADP-ribose); IP, immunoprecipitation; IR, irradiation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. Olaparib treatment promotes the autophosphorylation of DNA-PKcs. (A) The effect of olaparib on the phosphorylation of DNA-PKcs in response to IR. HeLa cells were treated with 10 uM olaparib for 24 h and irradiated with 8 Gy IR. After 1, 2, 4 and 8 h, the expression of phosphorylated DNA-PKcsSer2056 and DNA-PKcsThr2609 was detected by western blotting. (B) HeLa cells were treated with 1 or 10 uM olaparib for 24 h, and the cells were irradiated with 8 Gy IR. After 1 h, the expression of phosphorylated DNA-PKcs/S2056 was detected by western blotting. The effects of olaparib on the formation of gammaH2AX and DNA-PKcs/S2056 foci were detected by immunofluorescence staining in 8 Gy-irradiated HeLa cells. (C) The dynamic changes in gammaH2AX foci in 8 Gy-irradiated HeLa cells (magnification x100). (D) The dynamic changes in gammaH2AX foci in 8 Gy-irradiated HeLa cells. (E) The dynamic changes in DNA-PKcs/S2056 foci in 8 Gy-irradiated HeLa cells. *P