Edta free

RTMs isolated from 5–10 mice were washed three times with PBS and then incubated for 30 min at 37°C with 1.0 µm carboxylated magnetic beads (Dynabeads; Thermo Fisher Scientific) diluted at 1:20 ratio by complete DMEM/F-12 medium. Cells were then washed with ice-cold PBS and scraped, and cell pellets were washed two times with ice-cold PBS and once with hypotonic lysis buffer (250 mM sucrose/3 mM imidazole, pH 7.4). Cells were lysed in hypotonic lysis buffer containing inhibitors of phosphatases and proteases (EDTA-free; Thermo Fisher Scientific) and 250 U/ml of nuclease (Thermo Fisher Scientific) by 15 strokes in a Dounce homogenizer. Tubes with lysates were then transferred into a magnetic holder, and supernatants were removed. Phagosomes were washed four times with ice-cold PBS and centrifuged (13,000g, 5 min, 4°C) after the final wash. The supernatant was discarded, and pelleted phagosomes were stored at −80°C.

Cells were washed with cold PBS and resuspended in lysis buffer containing 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, in the presence of HaltProtease Inhibitor Cocktail, EDTA-Free (Thermo) and incubated on ice for 20 min. Lysates were sonicated for 10 min at 40 kHz in a bath sonicator (Emag Emmi-D280) and centrifuged at 18,000×g for 10 min at 4 °C. Total protein concentration was determined using Roti-Quant kit (Carl Roth) according to the manufacturer’s instructions. Lysates were diluted in Roti-Load (Carl Roth) and heated at 95 °C for 5 min. Then, 15–35 µg protein was loaded and separated by SDS-PAGE in 4–20% Mini-Protean TGX Precast gels (Bio-Rad). Gels were transferred to PVDF membranes using the iBlot system (Thermo). Membranes were blocked for 1 h in 5% low-fat powdered milk/1% Tween/TBS (TBS-T), washed and incubated overnight in primary antibodies at 4 °C and for 1 h at RT in HRP-conjugated secondary antibodies, all diluted in 3% milk/TBS-T. Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and the ImageQuant LAS 4000 mini. Tubulin was used as an internal loading control, and the relative intensities of protein bands were quantified using ImageJ and Excel.

The 10-day-old transgenic Arabidopsis seedlings expressing SOQ1-FLAG were used to extract total proteins with 1 ml IP buffer [10 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, and 1 pierce of protease inhibitor EDTA-free (Thermo)]. Then, 30 μl anti-FLAG M2 affinity gel (Cat# A2220; RRID: AB_10063035, Sigma) was added and incubated for 3 h at 4 °C. After washing three times with ice-cold IP buffer, the bound proteins were competitively eluted by 3× Flag Peptide (Beyotime). The presence of SOQ1-FLAG and HHL1 in the eluate (IP fraction) and input fraction was analyzed by SDS-PAGE and immunoblotting with anti-HHL1 (17 (link)) or HRP-conjugated anti-FLAG (Cat.#A8592; RRID: AB_439702, Sigma) antibodies.
The protoplasts from 25-day-old transgenic Arabidopsis plants expressing SOQ1-FLAG were cotransformed with HHL1-nYFP or HHL1-NVWA-nYFP, and the Co-IP assay was performed as described above. Anti-LCNP (Cat.#PAB211040, Orizymes), anti-MYC (Cat.#HT101, TransGen Biotech;), and anti-HA (Cat.#H3663, Sigma) antibodies were used.