5 bromo 4 chloro 3 indolyl phosphate

Total protein was prepared from leaves and roots of plants grown for 2 weeks on GM agar medium (Valvekens et al., 1988 (link)). Tissues were ground under liquid nitrogen and homogenized in the extraction buffer [50 mM Tris-MES (pH 7.5), 300 mM sucrose, 150 mM NaCl, 10 mM CH₃COOK, 5 mM EDTA, 20 μM leupeptine, 100 μM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM phenylmethylsulfonyl fluoride]. The lysate was centrifuged at 10,000 g for 15 min, and the supernatant was collected. Protein concentrations were determined using a Bio-Rad protein assaying kit (Bio-Rad) based on the Bradford method (Bradford, 1976 (link)), using bovine serum albumin as a standard. Proteins were separated in 10% (w/v) polyacrylamide gel, and transferred to Immobilon-P membrane (Millipore) by electroblotting. Ten micrograms of crude proteins were loaded to each lane of the gels. The blot was incubated with anti-GFP mouse monoclonal antibody (Nacalai Tesque, Japan), followed by incubation with goat anti-mouse IgG conjugated to alkaline phosphatase (Promega). The presence of immuno-reactive protein was detected through the use of 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (Promega).

C. glutamicum strains W, GAD, and GADΔpknG were cultured in test tubes at 30°C for 24 hours in 5 mL BHI medium containing 25 μg mL⁻¹ kanamycin. Each culture (0.2 mL) was transferred to 20 mL BHI medium containing 25 μg mL⁻¹ kanamycin in a 200 mL shaker flask. After fermentation for 24 hours, the cells from a 1 mL culture were centrifuged at 8,000 × g for 5 min, washed once in 50 mM Tris–HCl (pH 6.8) buffer, suspended in 1 mL of this buffer, and then 0.7 g of 0.1-mm diameter glass beads YGB01 (Yasui Kikai, Japan) was added to the tube. The cells were disrupted using a Shake Master Neo (Bio Medical Science) by shaking the tube three times at 1,500 rpm for 1 min at 1-min intervals. After centrifugation at 9,000 × g for 5 min, the supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The separated proteins were electroblotted onto a polyvinylidene fluoride membrane (Millipore, Boston, MA, USA) and then reacted sequentially with a mouse anti-FLAG M2 monoclonal antibody (Sigma, St. Louis, MO, USA) and a goat anti-mouse IgG alkaline phosphate conjugate (Promega) secondary antibody. The membrane was stained with 4-nitro-blue tetrazolium chloride (Promega) and 5-bromo-4-chloro-3-indolyl phosphate (Promega) according to the manufacturer’s instructions.

Total proteins from different tissues of male and female beetles, including antennae, head (without antennae), thorax, abdomen, leg, and wing tissues, were extracted and standardized to 7.5 μg per sample. After electrophoretic separation, protein bands were trans-ferred from a 15% SDS-PAGE to a nitrocellulose membrane (0.2 μm, Millipore, United States), as described by Kyhse-Andersen (1984) (link). After treatment with 0.2% non-fat dry milk and 0.05% Tween-20 in PBS overnight, the nitrocellulose membrane was then incubated with the primary antiserum obtained previously at a dilution of 1:5000 based on the ELASA result in Supplementary Figure S1. Goat anti-rabbit IgG- horseradish peroxi-dase conjugate (diluted by 1:1000; Fermentas, MD, United States) was used as the secondary antibody. Immunoreactions were visualized by adding 5-bromo-4-chloro-3- indolyl-phosphate and 4-chloro-1-naphthol (Promega, WI, United States).