Anti ATM, phospho Ser1981  

Rockland's anti-ATM pS1981 mouse monoclonal antibody (Catalog # 200-301-400) detects ATM phosphorylated on Ser 1981 by Indirect immunofluorescence microscopy. Shown are hTCEpi cells (courtesy of Dr. Danielle Robertson) infected with HSV-1 at MOI 5.0 and fixed at 8 hpi with 3% paraformaldehyde/2% sucrose for 10 min. After rinsing, cells were permeabilized with 0.5% Triton X-100 for 5 min, blocked with 3% BSA for 30 min, and stained with Rockland's primary anti-ATM pS1981 antibody overnight at 5 μg/mL (1:200). Secondary staining was performed with Alexa Fluor 594 anti-mouse antibody. Images were taken with Olympus AX70 compound epifluorescence microscope equipped with Spot RT Slider camera. Experiment was performed by Oleg Alekseev in the laboratory of Dr. Jane Azizkhan-Clifford at Drexel University College of Medicine.

Western Blot of Rockland's Protein A Purified Mab anti-ATM Protein Kinase pS1981. Lane 1: HEK293 cells treated with doxorubicin pre-incubation of peptide with 50 μg of immunizing phospho peptide negates specific staining. Lane 2: HEK293 cells treated with doxorubicin. A 370 kDa band corresponding to phosphorylated ATM is detected (lane 2). The lysate was prepared with HALT phosphatase inhibitor (Pierce). Load: ~30μg. Primary antibody: anti-ATM Protein Kinase pS1981 diluted 1:500 overnight at 4°C. Secondary Antibody: IRDye？800 conjugated Gt-a-Mouse IgG [H&L] (code 610-132-121) at 1:10,000 for 40 min at room temperature. LICOR's OdysseyR Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.

Anti ATM Antibody showing overlay of anti-ATM pS1981 staining. Cells were fixed 15 min after 5 Gy (IR+) of irradiation, then labeled with antibody. See Kitagawa et al. for additional details.

Anti ATM Antibody showing overlay of anti-ATM pS1981 staining. Cells were fixed 15 min after 5 Gy (IR+) of irradiation, then labeled with antibody. See Kitagawa et al. for additional details.

Anti ATM Mab with human derived HEK293 cells treated with doxorubicin using Rockland's Protein A Purified Mab anti-ATM Protein Kinase pS1981(clone 10H11.E12). A 370 kDa band corresponding to phosphorylated ATM is detected (arrowhead, lane 2). The lysate was prepared with HALT phosphatase inhibitor (Pierce). Pre-incubation of peptide with 50 μg of immunizing phospho peptide negates specific staining (lane 1). Approximately 30 μg of lysate was added to each lane of an SDS-PAGE gel under non-reducing conditions. The protein was transferred to nitrocellulose using standard methods. After blocking the membrane was probed with the primary antibody diluted 1:500 overnight at 4°C followed by washes and reaction with a 1:10,000 dilution of IRDye？800 conjugated Gt-a-Mouse IgG [H&L] (code 610-132-121) for 40 min at room temperature. LICOR's OdysseyR Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.