Mouse monoclonal antibody against mta1

For co-localization analysis, HCT116 cells were plated on sterilized coverslips, rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Then the cells were permeabilized with 0.25% Triton X-100 in PBS at room temperature for 10 min. After an incubation with blocking buffer (0.5% bovine serum albumin in PBS) for 30 min, the coverslips were simultaneously co-incubated with two primary antibodies: a mouse monoclonal antibody against MTA1 (Abcam) and a rabbit polyclonal antibody against Ku70 (Santa Cruz) or Ku80 (Santa Cruz, USA) over night at 4 °C. After washing with PBS, cells were then co-incubated with a FITC-conjugated goat anti-mouse antibody (ZSGB-BIO) and a TRITC-conjugated goat anti-rabbit antibody (ZSGB-BIO) for 1 h at room temperature. The coverslips were then mounted with mounting medium containing DAPI (ZSGB-BIO). And the fluorescent images were acquired using a fluorescent microscope (Leica).

For co-IP analysis, 1 mg of cell lysate was prepared and incubated overnight with 2 μg antibody at 4 °C on a rotator, followed by an incubation with 25 μl protein A/G agarose beads (Santa Cruz) at 4 °C for 2 h. The immunoprecipitates were washed with NP-40 buffer (50 mM Tris–HCl, pH 8.0, 0.5% NP-40, 10% glycerol, 150 mM NaCl, 2 mM MgCl₂, and 1 mM EDTA) for at least 3 times and collected after centrifugation at 6000 rpm for 5 min. The beads were then boiled in 2× loading buffer for 10 min and the supernatants were collected for subsequent western blot analysis. The antibodies used: mouse monoclonal antibody against MTA1 (Abcam), rabbit polyclonal antibody against Ku70 (Santa Cruz), rabbit polyclonal antibody against Ku80 (Santa Cruz) and Rabbit IgG (Santa Cruz).