Phospho atm ser1981

Protein extraction and Western blotting were performed as described by Santra et al. [25] (link). Briefly, cells were washed twice with ice-cold PBS, harvested, and collected by centrifugation. Protein extracts were prepared by lysing the cells in lysis buffer containing 50 mM Tris pH 7.4, 200 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 0.5% Triton X-100, and protease inhibitor cocktail on ice for 20 minutes [25] (link). Equal amounts of total protein were separated using SDS-PAGE and transferred onto the PVDF membranes (Millipore). Blots were developed by chemiluminescence method using LAS 4000 Images (GE). Densitometry analysis of the immunoblot was performed using the Image J software.
Antibody against the α-tubulin, β-actin, and anti-FLAG antibody was obtained from Sigma. Antibodies against the SDS22, GFP, PARP1, phospho AKT(S473), phospho AKT(T308), AKT, PARP1, MEK, ERK, p-cJUN, Twist, Snail, p53, APAF-1, Bcl2, BAX ATM, and AKT were obtained from Santa Cruz Biotechnology (Santacruz, CA). E-cadherin was obtained from BD Biosciences (USA).c-Myc, phospho MEK (Ser217/221), phospho ERK (Thr202/Tyr204), PUMA, BCL-XL, BAX, Caspase 9, and anti-mouse secondary antibody were obtained from Cell Signaling Technology, USA. Phospho-ATM Ser1981 was obtained from Rockland, USA.

Immunoblot analysis was carried out as described previously.46, 47 Antibodies specific for the following were used: MCM834; MCM934; Pan‐RAS (sc‐166691; Santa Cruz Biotechnology, Dallas, TX, USA); p53 (OP43; Calbiochem, Dallas, TX, USA); Chk1 (sc‐8408; Santa Cruz Biotechnology); phospho‐Chk1 Ser345 (#2348S; Cell Signaling Technology, Danvers, MA, USA); Chk2 (#05‐649; Upstate, Lake Placid, NY, USA); phospho‐Chk2 Thr68 (#2661; Cell Signaling Technology); phospho‐H2AX Ser139 (#07‐164; Millipore, Burlington, MA, USA); phospho‐ATM Ser1981 (#20772; Rockland, Limerick, PA, USA); and Rad51 (70‐012; Bio Academia, Osaka, Japan). The following secondary Abs were used: HRP‐rabbit anti‐mouse IgG (H + L) (61‐6520; Invitrogen, Carlsbad, CA, USA); HRP‐goat anti‐rabbit IgG (H + L) (65‐6120; Invitrogen); Alexa 594‐conjugated donkey anti‐rabbit IgG (H + L) (A21207; Invitrogen); and Alexa 488‐conjugated goat anti‐mouse IgG (H + L) (A11029; Molecular Probes, Eugene, OR, USA).