Anti flag m2 conjugated agarose

The rabbit polyclonal antibodies against human STING were described previously^{23 (link)}. Rabbit antibodies against mouse STING, p-IRF3(Ser396), p-TBK1(Ser172), p-IKKβ(Ser177), ATG5, ATG9, Beclin1, calreticulin, and GAPDH were from Cell Signaling. Mouse antibody against STING was purchased from R&D Systems; rabbit antibodies against human IRF3, TGN38, and ARF1 and mouse antibody against CD63 were from Santa Cruz Biotechnology; rabbit antibody against LC3 was from Novus Biologicals; mouse antibodies against P62 and GGA3 were from BD Transduction Laboratories; mouse antibody against ERGIC-53 was from Axxora; rabbit antibody against Sec22b was from Synaptic Systems; mouse antibody against Flag tag, rabbit antibodies against ERGIC53, and β-tubulin, anti-Flag (M2)-conjugated agarose and anti-HA–conjugated agarose were from Sigma; HA antibody were from Covance; rabbit antibodies against GBF1, LAMP2, and Giantin were from Abcam; rabbit antibody against beta-COP was from Thermo Fisher; rabbit antibodies against ARFGEF1, ARFGEF2, and SEC24C were from Bethyl Laboratories.

HEK293 cells stably expressing YFP-ASC and with intact TLR4/MyD88 signaling were seeded at 5 × 10⁵ cells/well in six-well plates. Cells were transfected with plasmids expressing Flag-tagged PKR (PKR cDNA ORF clone, human, N-DDK (Flag) tag, BioSite CA HG10080-NF) and GFP-tagged NLRP3 (pEGFP-C2-NLRP3, addgene CA 73955). Cells were lysed in lysis buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 1% Nonidet P-40 (NP-40), 2 mM EDTA). Ten microliters of anti-FLAG-M2-conjugated-agarose (Sigma) was pre-incubated with 5% BSA in PBS to minimize non-specific interaction before added to 100 μl of the cell lysate in 500 μl of IB solution [5 mM Tris-Cl, 10 mM HEPES, pH 7.5, 10% glycerol, 50 mM KCl, 0.05% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol, 1× complete protease inhibitor (Roche Diagnostics)]. Incubation was at 4°C with gentle rotation overnight after which the beads were collected, washed three times with 500 μl of IB solution for 10 min each. The beads were resuspended in 30 μl of 2X SDS sample buffer, boiled after which 20 μl of the elutates were subjected to Western blot using antibodies described above.