Anti ha 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.

The 10-day-old Pro35S:Flag-HA-SOS2 seedlings (about 5 g) in the sos2-2-mutant background^{21 (link)} grown on MS Petri dishes were collocated and ground to a fine powder in liquid nitrogen. The plant material was incubated in IP buffer for 30 min on ice (10 mM Tris, pH 7.5, 0.5% Nonidet P-40, 2 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1% protease inhibitor cocktail, Roche) and centrifuged at 12,000 × g at 4 °C for 10 min to remove cell debris. The anti-HA-conjugated agarose (Sigma-Aldrich) was used for HA-SOS2 immunoprecipitation. The SOS2 protein and its interacting proteins were then analyzed by a LTQ ORBITRAP Velos mass spectrometer (ThermoFisher Scientific). Database searches were performed on an in-house Mascot server (Matrix Science Ltd.) against International Protein Index (IPI) Arabidopsis database. The matched peptides were filtered with 0.05 significance threshold. The Mascot Percolator scores were used for all peptides.
To confirm the phosphorylation site of SOS2^Ser294 site by PKS5, samples containing 15 µg of His-tagged SOS2 and His-tagged PKS5 retained on beads. Total proteins were incubated in 30 µL of kinase buffer (20 mM Tris–HCl, pH 8.0, 1 mM DTT, 5 mM MgCl₂, and 10 μM ATP) at 30 °C for 30 min, His-tagged PKS5 were removed by a 5-min centrifugation at 500 × g at 4 °C. His-tagged SOS2 were collected and analyzed by LC–MS/MS.