Protein a g agarose slurry

For immunoprecipitation, the spinal cord tissue was isolated from E12.5 C57Bl6 embryos and lysed in the RIPA-buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, Triton X-100 1 % (v/v), Na-Deoxycholat 0.5 % (w/v), all Sigma-Aldrich) with protease inhibitors: 1 mM phenylmethanesulfonylfluoride (PMSF) and 1 μg/ml Aprotinin, all Roche. 30 μl Protein A/G agarose slurry (Santa Cruz Biotechnology, Dallas, TX) was incubated with 2.5 μg goat anti-mouse IgG conjugated with Cy3 (Dianova, Hamburg, Germany) for 1 h on a rotating wheel in PBS, followed by incubation with 4 μg of mAb 11E2 mouse anti-LRP1 α-chain (Storck et al. 2016 (link)) or 4 μg of isotype control mAb actin (BD) for 2 h in 1 ml PBS/A. 1000 μg of total protein was incubated with pre-labeled agarose slurry in 500 μl of RIPA buffer overnight, washed gently 3 times. Before SDS-polyacrylamide gel electrophorese, samples were boiled for 5 min in loading buffer (60 mM Tris-HCl pH 6.8, 2.5% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.01 % bromophenol blue). 5750^LeX, 487^LeX and LRP1 α-chain epitopes were subsequently detected by Western blot analysis.

C57BL/6J wild type or NLRP3-deficient BMDMs were stimulated with LPS (1 μg/mL, 4 h) to induce upregulation of NLRP3. Cells were then incubated with 15d-PGJ2 or biotinylated 15d-PGJ2 (Cayman Chemical) (50 μM, 1 h). Sucrose buffer lysates were made in the presence of Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Approximately 200 μg of cell lysate was mixed with 5 μg of anti-NLRP3 antibody (MAB7578, R&D Systems) and placed on a rotary shaker at 4°C for 2 h. Protein A/G agarose slurry (20 μL, Santa Cruz Biotechnology) was added and the incubation was continued overnight. Bound immune complexes were washed with PBS four times prior to elution of proteins in 2X SDS loading buffer. Biotinylated adducts were detected with IR680-conjugated streptavidin (LI-COR Bioscienses) by Western blotting.