Journal: DNA Repair

Article Title: ATM protects against oxidative stress induced by oxidized low-density lipoprotein

doi: 10.1016/j.dnarep.2011.05.004

Figure Lengend Snippet: OxLDL induces phosphorylation of H2AX in ATM-deficient fibroblasts: AT22 and VA13 cells were treated (A) with 100 μg/ml oxLDL for 16 h or (B) with 100 μg/ml lipoprotein at indicated times. VA13 cells were treated with 1 μM staurosporine (St) for 4 h to induce phosphorylation of H2AX and apoptosis. Cells were lysed and protein lysates were subjected to Western Blot analysis following SDS-PAGE. Polyclonal anti-γ-H2AX was used as primary antibody. Membranes were then stripped and incubated with anti-PARP and anti-caspase-3 antibody as primary antibodies. β-Actin served as a loading control.

Article Snippet: The following primary antibodies were used: (i) polyclonal rabbit phospho-ATM antibody (recognizing human ATM phosphorylated at Ser 1981 , termed pATM, R&D Systems, Minneapolis, MN, USA); (ii) sequence-specific polyclonal rabbit anti-ATM antibodies raised against synthetic peptides corresponding to amino acids 819–844 (Calbiochem) or 2550–2600 (Abcam, Cambridge, U.K.) of human ATM; (iii) polyclonal rabbit anti-caspase-3 antibody (raised against full-length caspase-3, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (iv) polyclonal anti-β-tubulin (Santa Cruz Biotechnology); (v) polyclonal phospho-histone H2AX antibody (recognizing H2AX only when phosphorylated at Ser 139 , termed γ-H2AX, Cell Signaling Technology, Beverly, MA, USA); (vi) rabbit monoclonal anti-p21 Waf1/Cip1 antibody (12D1, Cell Signaling Technology, Beverly, MA, USA), (vii) monoclonal anti-β-actin antibody (Santa Cruz Biotechnology); (viii) monoclonal anti-Poly (ADP-ribose) polymerase (PARP) antibody (recognizing the 116 kDa and the 85 kDa apoptosis-related cleaved fragment, Biomol, Hamburg, Germany).

Techniques: Phospho-proteomics, Western Blot, SDS Page, Incubation, Control

Journal: DNA Repair

Article Title: ATM protects against oxidative stress induced by oxidized low-density lipoprotein

doi: 10.1016/j.dnarep.2011.05.004

Figure Lengend Snippet: ATM-kinase-dependent activation of p21 in VA13 fibroblasts by oxLDL: AT22 and VA13 cells were incubated with 100 μg/ml oxLDL at indicated times in the absence or presence of 10 μM ATM-I (preincubated for 1 h). Cell lysates were subjected to SDS-PAGE, proteins were transferred to nitrocellulose and membranes were incubated with anti-p21 Waf1/Cip1 as the primary antibody. β-Actin was used as loading control. One representative experiment out of two is shown.

Article Snippet: The following primary antibodies were used: (i) polyclonal rabbit phospho-ATM antibody (recognizing human ATM phosphorylated at Ser 1981 , termed pATM, R&D Systems, Minneapolis, MN, USA); (ii) sequence-specific polyclonal rabbit anti-ATM antibodies raised against synthetic peptides corresponding to amino acids 819–844 (Calbiochem) or 2550–2600 (Abcam, Cambridge, U.K.) of human ATM; (iii) polyclonal rabbit anti-caspase-3 antibody (raised against full-length caspase-3, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (iv) polyclonal anti-β-tubulin (Santa Cruz Biotechnology); (v) polyclonal phospho-histone H2AX antibody (recognizing H2AX only when phosphorylated at Ser 139 , termed γ-H2AX, Cell Signaling Technology, Beverly, MA, USA); (vi) rabbit monoclonal anti-p21 Waf1/Cip1 antibody (12D1, Cell Signaling Technology, Beverly, MA, USA), (vii) monoclonal anti-β-actin antibody (Santa Cruz Biotechnology); (viii) monoclonal anti-Poly (ADP-ribose) polymerase (PARP) antibody (recognizing the 116 kDa and the 85 kDa apoptosis-related cleaved fragment, Biomol, Hamburg, Germany).

Techniques: Activation Assay, Incubation, SDS Page

Journal: DNA Repair

Article Title: ATM protects against oxidative stress induced by oxidized low-density lipoprotein

doi: 10.1016/j.dnarep.2011.05.004

Figure Lengend Snippet: ATM-kinase activation and induction of p21 in endothelial cells by oxLDL: EA.hy926 cells were incubated with 100 μg/ml oxLDL (A, B, C) or LDL (B) at indicated times or for 90 min in the absence or presence of 10 μM (C) or 10 and 30 μM (B) ATM-I (preincubated for 1 h). As positive controls, cells were treated with 200 μM H 2 O 2 for 15 min; after medium exchange the cells were incubated for another 90 min (105*) (A). Cell lysates were subjected to SDS-PAGE, proteins were transferred to nitrocellulose and membranes were incubated with anti-pATM (A, B) or anti-p21 (C) as primary antibody. β-Tubulin (A, B) or β-actin (C) was used as loading control. One representative experiment out of two is shown. (D) Colony forming efficiency of EA.hy926 cells treated with 30 μg/ml lipoprotein in the absence or presence of 10 μM ATM-I was performed exactly as described in B. Data are given as number of colonies (as percentage compared to untreated cells, set as 100%). Values represent the means ± SD ( n = 3). UT (untreated cells). ** p < 0.01 and *** p < 0.001.

Article Snippet: The following primary antibodies were used: (i) polyclonal rabbit phospho-ATM antibody (recognizing human ATM phosphorylated at Ser 1981 , termed pATM, R&D Systems, Minneapolis, MN, USA); (ii) sequence-specific polyclonal rabbit anti-ATM antibodies raised against synthetic peptides corresponding to amino acids 819–844 (Calbiochem) or 2550–2600 (Abcam, Cambridge, U.K.) of human ATM; (iii) polyclonal rabbit anti-caspase-3 antibody (raised against full-length caspase-3, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (iv) polyclonal anti-β-tubulin (Santa Cruz Biotechnology); (v) polyclonal phospho-histone H2AX antibody (recognizing H2AX only when phosphorylated at Ser 139 , termed γ-H2AX, Cell Signaling Technology, Beverly, MA, USA); (vi) rabbit monoclonal anti-p21 Waf1/Cip1 antibody (12D1, Cell Signaling Technology, Beverly, MA, USA), (vii) monoclonal anti-β-actin antibody (Santa Cruz Biotechnology); (viii) monoclonal anti-Poly (ADP-ribose) polymerase (PARP) antibody (recognizing the 116 kDa and the 85 kDa apoptosis-related cleaved fragment, Biomol, Hamburg, Germany).

Techniques: Activation Assay, Incubation, SDS Page

Journal: DNA Repair

Article Title: ATM protects against oxidative stress induced by oxidized low-density lipoprotein

doi: 10.1016/j.dnarep.2011.05.004

Figure Lengend Snippet: OxLDL induces phosphorylation of H2AX in ATM-deficient fibroblasts: AT22 and VA13 cells were treated (A) with 100 μg/ml oxLDL for 16 h or (B) with 100 μg/ml lipoprotein at indicated times. VA13 cells were treated with 1 μM staurosporine (St) for 4 h to induce phosphorylation of H2AX and apoptosis. Cells were lysed and protein lysates were subjected to Western Blot analysis following SDS-PAGE. Polyclonal anti-γ-H2AX was used as primary antibody. Membranes were then stripped and incubated with anti-PARP and anti-caspase-3 antibody as primary antibodies. β-Actin served as a loading control.

Article Snippet: The following primary antibodies were used: (i) polyclonal rabbit phospho-ATM antibody (recognizing human ATM phosphorylated at Ser 1981 , termed pATM, R&D Systems, Minneapolis, MN, USA); (ii) sequence-specific polyclonal rabbit anti-ATM antibodies raised against synthetic peptides corresponding to amino acids 819–844 (Calbiochem) or 2550–2600 (Abcam, Cambridge, U.K.) of human ATM; (iii) polyclonal rabbit anti-caspase-3 antibody (raised against full-length caspase-3, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (iv) polyclonal anti-β-tubulin (Santa Cruz Biotechnology); (v) polyclonal phospho-histone H2AX antibody (recognizing H2AX only when phosphorylated at Ser 139 , termed γ-H2AX, Cell Signaling Technology, Beverly, MA, USA); (vi) rabbit monoclonal anti-p21 Waf1/Cip1 antibody (12D1, Cell Signaling Technology, Beverly, MA, USA), (vii) monoclonal anti-β-actin antibody (Santa Cruz Biotechnology); (viii) monoclonal anti-Poly (ADP-ribose) polymerase (PARP) antibody (recognizing the 116 kDa and the 85 kDa apoptosis-related cleaved fragment, Biomol, Hamburg, Germany).

Techniques: Phospho-proteomics, Western Blot, SDS Page, Incubation, Control