Protein a mag sepharose xtra

Co-immunoprecipitation and RT-PCR were combined to examine the association of viral RNA with ZAP proteins. Cells were lysed by RIPA lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate) containing protease inhibitor cocktail and RNase inhibitor. For RIP assay using antibody against endogenous ZAP-L/S, mock and virus-infected A549 cell extracts were immunoprecipitated with preincubated mixture of protein A (Protein A Mag Sepharose Xtra, 28-9670-62, GE Healthcare) with anti-ZC3HAV1 (ZAP) antibody (16820-1-AP, Proteintech) or IgG (12–370, EMD Millipore) at 4°C for 4 h. For RIP assay using anti-V5 antibody, extracts of virus-infected 293T/17 cells expressing EGFP (without V5-tag), ZAP-L-V5, and ZAP-S-V5 (WT and del4zFs) were incubated with anti-V5 agarose affinity gel (A7345, Sigma-Aldrich) at 4°C overnight. The antibody-protein-RNA complex was then washed five times by RIPA lysis buffer, and the bound RNA was purified by RNeasy Mini Kit (Qiagen). RT-PCR was performed by use of the 3′-UTR specific primers (S1 Table).

For the pull-down experiment, purified COI1-GST (5 nM), OG-conjugated JAZ peptide (10 nM), and coronatine analogs (100 nM) in 500 μL of incubation buffer (50 mM Tris-HCl, pH 7.8, containing 100 mM NaCl, 10% glycerol, 0.1% Tween20, 20 mM 2-mercaptoethanol, and 100 nM IP₅)^{10 (link),17 ,30 (link),62} were combined with anti-fluorescein antibody (Abcam, ab19491, 0.25 μL) and incubated for 10–15 h at 4 °C with rotation. After incubation, the samples were combined with Protein A Mag Sepharose Xtra (GE Healthcare, 25 µL in 50% incubation buffer slurry). After 3 h incubation at 4 °C with rotation, the samples were washed three times with 500 µL of fresh incubation buffer. The washed beads were resuspended in 50 µL of SDS-PAGE loading buffer containing dithiothreitol (DTT, 100 mM). After heating for 10 min at 60 °C, the samples were subjected to SDS-PAGE and analyzed by western blotting. The bound COI1-GST proteins was detected using anti-GST HRP conjugate (RPN1236, GE Healthcare, 2500-fold dilution in skimmed milk solution). Uncropped blot of Fig. 2c were shown as Supplementary Fig. 19. Uncropped blot of Fig. 2d,e and Supplementary Fig. 3 were shown as Supplementary Fig. 20. Uncropped blot of Fig. 3e were shown as Supplementary Fig. 21. Uncropped blot of Supplementary Fig. 4a were shown as Supplementary Fig. 22. Uncropped blot of Supplementary Fig. 6 were shown as Supplementary Fig. 23.