Ab19491

The primary antibodies used to detect specific proteins were as follows: mouse anti-SF1 antibody (2E12, Abnova H00007536-M01A) for CLIP, IP, IF and WB; rabbit anti-SF1 (Aviva ARP41214_T100) for IF and WB; anti-GAPDH (Millipore #MAB374), antitubulin (Abcam # ab7291), and anti-RFP (MBL #PM005, Japan) for WB; anti-GFP (Roche #1814460) for IP and WB; anti-FLAG M2 (Sigma #F1804) as a mock control for CLIP and IP; mouse anti-DIG (21H8, Abcam #ab420) and rabbit antifluorescein (Abcam #ab19491) for FISH. The secondary antibodies used were as follows: anti-mouse IgG HRP (GE Healthcare #NA931) for ELISA and WB; anti-rabbit IgG HRP (GE Healthcare #NA934) for WB; anti-mouse IgG Cy3 (Millipore #AP124C) and anti-rabbit IgG Alexa Fluor 488 (Life Technologies #A11008) for FISH and IF; and anti-DIG AP (Roche #1093274) and antifluorescein AP (Roche #1426338) for NB.

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.