Gtx70228

Cells were grown on coverslips, fixed in −20 °C 100% methanol for 10 min on ice, and permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT). Cells were incubated with blocking buffer (1% BSA, 0.1% Tween 20 in PBS) for 1 h at RT. Coverslips were incubated with primary antibodies for 1 h at RT, washed three times with 1% PBS and once with blocking buffer, and incubated with the appropriate fluorescent secondary antibody for 1 h at RT. The coverslips were then washed three times with 1% PBS and mounted on microscopy slides using mounting reagent with DAPI (Ibidi GmbH, Gräfelfing, Germany). Staining was analyzed using a Leica DMi8 inverted microscope with a 63x oil-immersion objective using the same laser parameters. All microscope images were analyzed with ImageJ software. Primary antibodies used: anti-RAD50 mouse monoclonal antibody (GTX70228, GeneTex), anti-NBS1 rabbit monoclonal antibody (#14956, Cell Signaling Technology), anti-MRE11 mouse monoclonal antibody (sc-135992, Santa Cruz Biotechnology), anti-p62 rabbit polyclonal antibody (NBP1-49955, Novus Biologicals), anti-LC3 rabbit polyclonal antibody (NP100-2220, Novus Biologicals) and anti-LAMP1 mouse monoclonal antibody (sc-20011, Santa Cruz Biotechnology).

Production of GST fusion proteins and GST-pulldown assays were performed as previously described [15 (link),16 (link)]. Endogenous co-immunoprecipitation assays were performed using PEL cell lines (1x10^7 cells/condition) harvested with TBS-T buffer (20 mM TRIS-HCl pH 7.4, 150 mM NaCl, 50 mM MgCl₂, 1% TritonX-100). Benzonase nuclease (Merck Millipore, 71205–3) was added to whole cell lysates (50U each 2x10⁶ cells) for 30 minutes at RT to digest nucleic acids. Subsequently, the samples were centrifuged at 20800 g for 10 minutes at +4°C and the supernatants used for immunoprecipitation. The input control corresponds to 4% of the lysate used for an individual immunoprecipitation sample. Protein G sepharose beads (GE Healthcare) were washed with TBS-T buffer and incubated for 5 hours at +4°C with α-LANA (rat, from ABI, 18-210-100) or α-Rad50 (mouse, from GeneTex, GTX70228) or negative control (α-IgG rat or α-IgG mouse) antibody. Finally, antibody-coupled-beads were washed with TBS-T buffer, resuspended in PBS and used immediately or stored shortly at +4°C.