Gfp trap beads slurry

GFP-trap co-immunoprecipitation were performed as in ref. ^{61 (link)}. In brief, a confluent 15 cm dish of RPE1 TP53^−/−BRCA1^−/− cells rescued or not with GFP-tagged BRCA1 wild type or I26A mutant were scrapped, washed in ice-cold PBS and lysed in 1 mL of ice-cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.5% Triton X-100, EDTA-free protease inhibitors (Roche) and 20 mM N-Ethylmaleimide (NEM)). Lysates were vortexed and incubated for 1 h at 4 °C while rotating with 500 Units of Benzonase (Millipore). Afterwards, samples were centrifuged for 1 h at 20,000 × g at 4 °C. As inputs, 20 µL of supernatants were saved per sample and the rest was incubated with 25 µL of GFP-Trap beads slurry (Chromotek) for 90 min at 4 °C while rotating. Subsequently, beads were washed three times with wash buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl₂, EDTA-free protease inhibitors (Roche), and 20 mM N-Ethylmaleimide (NEM)) and resuspended in LDS sample buffer 1X for immunoblotting procedures.

Brains and imaginal discs of 100 third instar larvae were dissected and squashed into 500 μl of lysis buffer: 50 mM Tris; 150 mM NaCl; 50 mM Sucrose; 5 mM EDTA; 5 mM ATP; 1 mM DTT; 0.3% Triton X-100; pH 7.5 and protease inhibitors (Complete mini tablets Roche # 05892791001). The extract was then incubated 40 min at 4 °C on a rotating wheel, and cellular debris were cleared by centrifugation (16,000g, 10 min at 4 °C). Twenty-five microlitre of GFP-Trap beads slurry (Chromotek # 090703001A) were equilibrated with Triton-free lysis buffer and incubated with the cleared extract. Immunoprecipitation was performed during 2 h at 4 °C with mild agitation. Beads were then washed three times with lysis buffer, two times with Triton-free lysis buffer and finally resuspended in 40 μl Laemmli sample buffer. Samples were further processed for SDS–PAGE and western blot.
For mass spectrometry, immunoprecipitates were processed by SDS–PAGE on 10% Precise Precast Protein Gels (Thermo Scientific). Bands were cut from the gel, and further processed for mass spectrometry using the NanoLC-ESI-MS/MS technique.

Five microlitres of GFP-Trap beads slurry (ChromoTek, Munich, Germany) were mixed with a 5 μM solution of GFP-fused bait proteins (GFP-LC3B or GFP-GABARAP) and incubated on a rotating wheel at 4 °C for 1 h. Subsequently the beads were washed twice with 150 mM NaCl, 50 mM Tris at pH 7.4, and incubated with 20 μg of prey solution (GST-Cx43^WT_NT or GST-Cx43^W4A+L7A_NT). Precipitates were analysed using Western blot using goat polyclonal antibodies against GST.