Complete mini edta free protease inhibitor cocktail

For immunoprecipitation, cells were lysed in RIPA buffer (Sigma) supplemented with Complete Mini EDTA-free Protease Inhibitor cocktail (Roche, Basel, Switzerland) and PhosStop (Roche). About 200 μg of lysates were pre-cleared for 30 min using normal mouse or rabbit IgG (Promega) and 20 μl protein A/G beads (Santa Cruz Biotechnology) prior to incubation with agarose beads conjugated to antibodies recognizing Flag- or Myc-conjugated beads (Sigma, 15 μl). Immunoblotting was performed as described previously .
About 100 ng of indicated recombinant proteins (Figures 6e and f) were incubated in phosphate-buffered saline supplemented with Complete Mini EDTA-free Protease Inhibitor cocktail (Roche), 0.05% Triton X100, 100 mm of NaCl and 0.05% bovine serum albumin for 1 h at 4°C. About 20 μl of DO1 (p53 antibody) conjugated to agarose (Santa Cruz Biotechnology, sc-126 AC) or 20 μl of LZAP custom antiserum conjugated to Protein A/G agarose (Santa Cruz Biotechnology) were added and incubated overnight at 40°C. The beads were spun down at 3000 r.p.m. for 1 min, washed four times with phosphate-buffered saline supplemented with 0.05% Triton X100 and 100 mm of NaCl, resuspended in 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and boiled for 3 min. Immunoblotting was performed as described.^{70 (link)}

Cytokine profiling was carried out using MesoScale Discovery V-PLEX Proinflammatory Cytokine Panel 1 (Human) ELISA kits, following the manufacturer’s instructions. Supernatants from LPS-stimulated and unstimulated cultures were diluted 1:300 and 1:5, respectively. Three parallel wells as technical replicates were measured per single data point. Where possible, data were normalised to total cell number as measured by high-content imaging. Otherwise, they were normalised to total cell protein content as measured by Pierce BCA assay (Thermo Fisher Scientific), having been washed once with sterile D-PBS and lysed in 60 μl RIPA buffer supplemented with Roche cOmplete Mini EDTA-free protease inhibitor cocktail.

Cytokine profiling was carried out using MesoScale Discovery V-PLEX Proinflammatory Cytokine Panel 1 (Human) ELISA kits, following the manufacturer’s instructions. Supernatants from LPS-stimulated and unstimulated cultures were diluted 1:300 and 1:5, respectively. Three parallel wells as technical replicates were measured per single data-point. Data were normalised to total cell protein content as measured by Pierce BCA assay (Thermo Fisher Scientific), having been washed once with sterile D-PBS and lysed in 60 µl RIPA buffer supplemented with Roche cOmplete Mini EDTA-free protease inhibitor cocktail.