Horseradish peroxidase hrp conjugated anti rabbit igg

Cell lysates were obtained separated by 10% sodium dodecyl sulfate poly-acrylamide gel electrophoresis under reducing conditions as previously described [25 (link)]. After electrophoresis, samples were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) [25 (link)]. Membranes were incubated in 5% dry milk TBS-T buffer for 1hr, with vimentin, E-cadherin or N-cadherin (1:1000) mAbs (R&D Biotechnologies, Minneapolis, MN, USA). Phospho^(Ser1981)-ATM mAb (10H11) (Thermo-Fisher Scientific, Waltham, MA, USA) and ATM mAb, (Invitrogen, Rockford, IL, USA) were used to detect ATM expression and activity. The membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000) (Jackson Immunoresearch, West Grove, PA, USA) in 5% dry milk and TBS-T for 1 h. Thereafter, blots were developed with the EZ-ECL chemiluminescence detection kit (Bilogical Industries, Beit-HaEmek, Israel) according to the manufacturer’s instructions. β-actin (1:500) (Santa Cruz Biotechnology, Dallas, TX, USA) was used for loading control [25 (link)].
ATM activity was assayed via IB of the phosphorylation products, as described using Phospho-ATM vs. ATM mAbs (1:500) [26 (link)].

ESCC cell lines ECA109 and TE13 were obtained from Shanghai Institute of Cell Biology (Shanghai, China) and were maintained in DMEM medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, GE Healthcare, Little Chalfont, UK), 1% penicillin/streptomycin (Invitrogen, Life Technologies). Cells were maintained in an incubator at 37 °C, in an atmosphere containing 5% CO₂. HUVECs were isolated from human umbilical cord veins using a standard procedure described previously27 (link), and grown in EBM-2 medium with SingleQuots™ (Lonza, Walkersville, MD, USA) containing VEGF and other growth factors.
Cells in the IR group were subjected to a 2, 4, 6 or 8 Gy of X-ray irradiation from a medical linear accelerator (Elekta Precise) at room temperature.
anti-CD31, anti-CD105, anti-CD31, anti-CD105, anti-lectin, anti-α-SMA, anti-VEGF, anti-HIF-1α (Abcam), fluorescein (FITC)-conjugated anti-mouse IgG, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA). rh-Endo was provided by Simcere Pharmaceuticals (Nanjing, China).