Anti act

The specific polyclonal anti-S2P2 antibodies were custom produced in rabbits by Davids Biotechnologie GmbH (Germany), using the highly specific synthetically obtained antigen (AA sequence: DNDPDSDIPVDDRNLLKNR). Other primary antibodies (anti-H3, anti-ACT, anti-COXII, anti-TOC75, anti-PsbA, anti-PsbC, anti-PsbD, and anti-Lhcbs) as well as secondary antibodies were purchased from Agrisera AB (Vannas, Sweden).

Subcellular fractionations were isolated using a rapid plant fractionation kit (Invent Biotechnologies, Plymouth, MA, USA). Briefly, the 7-day-old soybean seedlings were ground with buffer A; then, the sample was centrifugated at 1500× g for 5 min. For cytosolic fractions, the supernatant was transfer to a new tube and centrifuged at 4000× g for 10 min. The supernatant was transferred a new tube and centrifuged at 16,000× g for 30 min. The cytosolic fraction was obtained from the supernatant. For the nuclear fraction, the pellet of ground seedling was suspended with cold phosphate-buffered saline (PBS) and centrifuged at 1200× g for 5 min. The pellet resuspended with buffer B was centrifuged at 1200× g for 5 min, and the pellet resuspended with cold PBS was overlaid with buffer C followed by a centrifugation at 1200× g for 5 min. The nuclear fraction was obtained from the well-washed pellet. For immunoblotting, anti-GmMPK6 (Ab frontier, Seoul, Korea), anti-HIS (Abcam, Waltham, MA, USA), anti-ACT (Agrisera, Vännäs, Sweden), and HRP-conjugated secondary antibody (Abcam) were used. The signals were detected using a Fusion SL (Vilber Lourmat, France). All experiments were performed in triplicates, unless otherwise specified, with consistent results. Representative image data are shown.

The total protein was extracted from the protoplasts using an extraction buffer (50 mM Tris-Base, 150 mM NaCl, 10 mM NaF, 10 mM Na₃Vo₄, 1x Complete, and 0.2% Triton X-100). The proteins were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. For immunoblotting, the primary antibodies anti-HA (Roche), anti-GFP (Abcam), and anti-ACT (Agrisera) were used (1:1000). Next, an HRP-conjugated secondary antibody (Abcam) was added (1:10,000). The signal was detected using an IR-image detector Odyssey (LI-COR, Lincoln USA).