Western blotting assay was carried out as a standard protocol. The following antibodies were used: CREB1 (Cell Signaling Technology, Beverly, MA, USA), P-CREB1 (Cell Signaling Technology), Nrf-1 (Santa Cruz, CA, USA) and ligase IV (Santa Cruz), respectively. The membrane was incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody (Cell Signaling Technology). GAPDH (Proteintech, Chicago, USA) served as a loading control. Protein bands were detected using an Enhanced Chemiluminescence Detection System (Millipore, Billerica, MA, USA).
Ligase 4
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Ligase IV is a DNA repair enzyme that catalyzes the formation of phosphodiester bonds between adjacent DNA fragments. It plays a crucial role in the non-homologous end joining (NHEJ) pathway, which is responsible for repairing double-stranded breaks in DNA. Ligase IV is essential for maintaining genome integrity and cellular function.
Automatically generated - may contain errors
Lab products found in correlation
Nrf 1, by Santa Cruz Biotechnology (2 mentions)
Creb1, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection system, by Merck Group (1 mentions)
P creb1, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
P creb1, by Merck Group (1 mentions)
γh2ax, by Merck Group (1 mentions)
β catenin, by BD (1 mentions)
3 protocols using ligase 4
1
Western Blot Analysis of CREB, Nrf-1, and Ligase IV
2
Immunofluorescence Analysis of DNA Repair Proteins
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Retinal slides were fixed with 4% paraformaldehyde at room temperature for 20 min and subsequently incubated with 0.5% Triton X-100 for 10 min. Then, the samples were treated with a blocking solution (5% normal goat serum and 2% bovine serum albumin in PBS) for 30 min to prevent nonspecific antibody–antigen binding. γ-H2AX, ligase IV, Nrf-1, CREB1 and P-CREB1 expression was detected using the antibody of γ-H2AX (Millipore), ligase IV (Santa Cruz), CREB1 (CST), P-CREB1 (CST) and Nrf-1 (Santa Cruz). For the fluorescence visualization of antibody reactions, the primary antibodies were detected using secondary antibodies labeled with the fluorochromes Alexa Fluor 555 or 488 (CST), while the nuclei were detected with DAPI.
3
Western Blot Analysis of Cell Signaling
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Cells were lysed with radioimmunoprecipitation assay buffer containing protease inhibitor cocktail. Next, western blotting was performed using a standard protocol. The following primary antibodies were used overnight at 4°C: ligase IV (1:300; Santa Cruz Biotechnology, Dallas, TX, USA), GSK-3β (1:1000; CST), p-GSK-3β (1:1000; CST), β-catenin (1:2000; BD Biosciences, Franklin Lakes, NJ, USA), CREB1 (1:1000; CST), and p-CREB1 (1:1000; Proteintech, Chicago, IL, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:10,000; CST) served as a loading control. Proteins were visualized following incubation with a horseradish peroxidase-conjugated anti-rabbit or -mouse IgG secondary antibody (1:10,000; CST) for 1 hour at room temperature, and exposured by the enhanced ECL chemiluminescence system (Bio-Rad, Hercules, CA, USA). Relative intensities of bands were quantified by densitometry using ImageJ software (version 1.51k; National Institutes of Health, Bethesda, MD, USA) and normalized to GAPDH levels.
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