Activated charcoal

Ammonium was extracted and its concentration was determined with slight modifications to the method reported by Hachiya et al.^{42 (link)}. Shoots were harvested, immediately frozen with liquid N₂, and stored at −80 °C until use. Frozen samples were ground with a Multi-Beads Shocker (Yasui Kikai Corp.) using zirconia beads (diameter, 5 mm). One mL of 0.1 N HCl and 500 µL of chloroform were added to the frozen powder, followed by vortexing for 15 min. The mixture was centrifuged at 12,000 × g at 8 °C for 10 min. The aqueous phase was transferred to a microtube containing 50 mg of acid-washed activated charcoal (No. 035-18081; Wako, Osaka, Japan). The mixture was vortexed and centrifuged at 20,400 × g at 8 °C for 10 min. The ammonium content of the supernatant was spectroscopically determined using an Ammonia Test Kit (No. 277-14401, Wako) according to the manufacturer’s instructions.

TTX and its analogs were extracted from the visceral part of fish with 0.1% acetic acid by heating in a boiling water bath for 10 min, according to the method for TTX described in Standard Methods of Analysis in Food Safety, Japan [37 ]. TTX and its analogs, 4,9-anhydroTTX and 5,6,11-trideoxyTTX, were isolated by successive column chromatography on activated charcoal (for chromatography; Wako, Osaka, Japan), Bio-Gel P-2 (200–400 mesh; Bio-Rad, Hercules, CA, USA), and Bio-Rex 70 (200–400 mesh; Bio-Rad, Hercules, CA, USA) according to the method described by Nagashima et al., [14 (link)]. 4,9-anhydroTTX thus isolated was used to prepare the antigen. In addition, an aliquot of isolated TTX (ca. 10 µmol) was dissolved in 5 mL of 0.1% acetic acid and heated in boiling water for 30 min. 4-epiTTX and 4,9-anhydroTTX produced in the reaction mixture were isolated by Bio-Rex 70 column chromatography (200–400 mesh, 1.5 × 115 cm) and used as the materials for the examination of their cross-reactions with the polyclonal antibody against TTX, which was produced in the present study as described below. Moreover, 11-oxoTTX was prepared from the isolated TTX by treating with the FeSO₄ and H₂O₂ mixture, according to Wu et al. [40 (link)] (Figure S1).

FCS and fatty acid–free BSA were incubated with activated charcoal (Wako, Japan) to remove all lipid fractions. The spleen and LNs were collected, and a single-cell suspension was prepared in RPMI1640 containing 1% FCS. T cells were negatively sorted and starved in serum-free medium (0.1% BSA/RPMI1640) for 2 hr at 37°C. Next, cells (1 x 10⁷) were stimulated with 1 μM LPA at 37°C for 2 or 5 min. The cells were collected by centrifugation and immediately dissolved in lysis buffer. The GTP-bound form of RhoA in the lysate was pulled down with Rhotekin and detected by western blotting using the RhoA Activation Assay Biochem Kit (Cytoskeleton, Denver, CO).