Gfp trap gtma 20

Reagents and chemicals used were LLOMe (L7393; Sigma-Aldrich), GPN (SC-252858; Scbt), BafA1 (1334; Tocris), PP242 (4257; Tocris), Monensin (M5273; Sigma-Aldrich), human serum (H2918; Sigma-Aldrich), Zymosan (Z4250; Sigma-Aldrich), murine IFNγ (315-05; Peprotech), DAPI (D9542; Sigma-Aldrich), IN-1 (17392; Caymen Chemical), MLi2 (5756; Tocris), and saliphenylhalamide (SaliP), and TRPML1 agonist C8 and ATG7 inhibitor ATG7-IN-3 were kindly provided by Casma Therapeutics. BAPTA-AM (B1205; Thermo Fisher Scientific), nocodazole (M1404; Sigma-Aldrich), GFP-Trap (gtma-20), and control magnetic agarose beads (bmab-20) were obtained from Chromotek.

To generate pCsVMV:PRRs-HA-1300 constructs, full-length PRR9, PRR7, and PRR5 coding sequence were amplified and inserted into the vector of pCsVMV:HA-1300. Fragments containing the ORFs of BBX18 and BBX19 were separately inserted into pCsVMV:GFP-1300 and 2 × 35S:FLAG-1307 vectors. All primer sequences are listed in Supplemental Table S7. The combinations of Agrobacterium carrying the indicated vectors were co-infiltrated into the leaves of 5-week-old N. benthamiana, and the samples were collected after 3 days of infiltration. Protein extraction and immunoprecipitation assays were performed following a method described previously (Wang et al., 2013 (link)) using GFP-Trap (GTMA-20, ChromoTek) magnetic beads. The incubation was about 1 h at 4°C followed by washing four times with protein extraction buffer using a magnetic stand. For immunoblot detection, GFP antibody (Cat#ab6556, Abcam), HA antibody (Cat#11867423001, Roche), and FLAG antibody (Cat#M20008M, Abmart) were used to detect the tagged proteins.