Protein a g plus agarose

Cells seeded in a 6-well tissue culture-treated dish (VWR) were treated with arsenite for 2 hours and lysed in a non-denaturing lysis buffer [10 mM Tris-HCl, pH 7.5, 1 mM DTT, 2 mM MgCl₂, 50 mM KCl, 1% Triton X-100, 1X HALT Protease/ Phosphatase Inhibitor Cocktail (78446, ThermoFisher)] for 10 min on ice. Equal volumes of Buffer I (20 mM Tris-HCl, pH 7.5, 0.4 mM NaCl, 1 mM DTT, 1 mM EDTA, 1% Triton X-100, 20% glycerol, 2 mM Na₃VO₄) were added. Lysates were precleared by incubation with Protein A/G PLUS-Agarose (sc-2003, Santa Cruz) for 30 min at 4^oC with rotation. To capture protein complexes, the lysates were incubated with antibodies specific for ZBP1 (mAb, Zippy-1, AG-20B-0010-C100, Adipogen) or G3BP1 (mAb, 66486–1-Ig, Proteintech) overnight at 4^oC with rotation. Protein A/G PLUS-Agarose (sc-2003, Santa Cruz) was added to lysates, incubated for 90 min at 4^oC with rotation, and washed three times in Buffer I for 5 min. Proteins bound to agarose beads were released by the addition of 4x Laemmli (250 mM Tris, pH 6.8, 8% SDS, 50% glycerol, 0.04% bromophenol blue) with 50 mM DTT and boiling for 15 min at 95^oC. Western immunoblot analysis as described above was followed using the antibody NBP1–76854 (Novus Biologicals) to detect ZBP1.

Activated T cells were treated and stimulated as described earlier. The cells were then lysed with immunoprecipitation buffer [25mM Tris (pH 8.0), 150 mM NaCl, 1% Brij-97, 0.5% n-Octyl-β-D-glucopyranoside, 5 mM EDTA, 1 mM Na₃VO₄, and complete protease inhibitor tablets] and pre-cleared with protein A/G plus agarose (Santa Cruz). For WASp immunoprecipitation experiments, the lysates were incubated with protein A/G plus agarose with or without the WASp antibody (Santa Cruz) overnight at 4°C. In Rac1/CDC42 activation experiments, lysates were incubated with PAK-PBD glutathione-conjugated agarose beads (Cytoskeleton) overnight at 4°C. The samples were washed, eluted, and the bound proteins were detected by Western blotting. Band intensity was quantified using Odyssey’s v3.0 software. To minimize variability between human donors, band intensities were further normalized to the band intensity of the two-minute timepoint for the ethanol treatment.

Proteins were extracted from MDA-MB-231 cells as described above. Protein concentrations were determined using the Bradford assay, and 500 μg lysate protein was brought to a final volume of 1 mL with PBS. Lysates were precleared with 1 μg of appropriate control IgG (Santa Cruz Biotechnology, sc-2763) and 20 μg of protein A/G PLUS-agarose (Santa Cruz Biotechnology, sc-2003) and kept on a rotator for 1 h at 4 °C. Lysates were centrifuged (500 ×g for 5 min at 4 °C) and 2 μg of GDH antibody (Santa Cruz Biotechnology, sc-160382) or corresponding IgG was added to the precleared lysates and kept on ice for 3 to 5 h. Following incubation, 30 μL of protein A/G PLUS-agarose was added to each tube and kept on a rotator overnight at 4 °C. Lysates were then centrifuged (500 ×g for 5 min at 4 °C). The pellet fractions were washed 4 times with PBS and then resuspended in 20 mL of loading buffer. Samples were electrophoresed on an 8% SDS-polyacrylamide gel and immunoblotted with an Acetylated-Lysine Mouse mAb (Ac-K-103) (Cell Signaling) antibody.