Sonicator

Paw soft tissue homogenization was performed in lysis buffer (20 mM Tris-base, 150 mM NaCl, 10% glycerol, 0.1% Triton-X-100, 1% Chaps, and 2 mM Ethylene glycol tetraacetic acid (EGTA)) with the addition of 1% protease inhibitor cocktail (v/v). Extracts were sonicated for 5 min (Fisher Scientific Sonicator) and incubated for 10 min on ice, and then were centrifuged at 12,500× g for 30 min at 4 °C. The obtained supernatants were stored immediately at −80 °C and used to evaluate immunoprecipitation and for Western blot analyses. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce).

An amount of 500 μL RIPA buffer for every 10 mg of tissue was added, homogenized thoroughly using a sonicator (Fisher Scientific, USA), and kept on ice for 30 min. Then, lysates were centrifuged in a microfuge at the 15,000 RPM speed for 20 min at 4 °C to pellet cell debris and were then transferred to the supernatant to a fresh microfuge tube without disturbing the pellet.
For each group, the protein concentration 10 µL of each sample was determined with a Bio-Rad expression and purification kit (Bio-Rad Laboratories, UK). Proteins were separated by gel electrophoresis SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% bovine serum albumin (BSA) for 2 h at room temperature to avoid non-specific binding. Then, the membranes were incubated with the indicated primary antibody, the rabbit polyclonal antibody, Cat. No. PA5-17848, 1:1000 dilution, for 24 h at 4 °C. The membranes were washed with PBS to remove any unbound primary antibody and incubated with goat anti-rabbit IgG, secondary antibody (Cat. No. A27036, 0.25 µg/mL, 1:4000 dilution) for 30 min at 4 °C, followed by three washes with PBS. β-actin was used as a control. Protein bands were visualized using the ChemiDoc MP Imaging System and Image Lab software (version 5.1).

β-D-galactosidase activity assays were performed as Tan SZ et al. described with minor modifications (Tan et al. 2018 (link)). Briefly, overnight, V. parahaemolyticus cultures were expanded 1:100 in 100 ml LB with or without appropriate amounts of inducers (IPTG and arabinose) and shaken at 37℃, 250 rpm. The cells were harvested at an OD₆₀₀ of ca. 0.8 and lysed in 5 ml PBS buffer with protease inhibitor cocktail (Roche Cat#11,697,498,001) using a Sonicator (Fisher) on ice-water mix. The lysates were centrifuged at 20,000 g for 30 min at 4℃ and supernatants were collected for activity assays. According to the manufacturer’s instructions, the total protein concentration was determined using a BCA protein assay kit (Beyotime, Beijing, Cat#P0011). Whole β-galactosidase activity assays of the lysates were determined by measuring the initial rate of the enzyme-catalyzed break of orthonitrophenyl-β-galactoside (ONPG). The absorbance change of OD₄₂₀ of reaction mixtures was measured, and the enzymatic activity was estimated as the rate of change of A₄₂₀ normalized by the total protein amount in the assays.

The cultured LAB was resuspended in distilled water at a concentration of 10 mg/ml and sonicated 5 seconds with a 10-second interval for 50 times on ice by using a sonicator (Fisher Scientific Co., Toronto, ON, USA) to make the homogeneous cell extracts. The suspension was centrifuged at 12,000 rpm for 15 min at 4 °C. And the resultant supernatants were filter-sterilized (pore size, 0.45 μm), lyophilized, and kept at −80 °C until use. The 3T3-L1 cells were treated with the concentrations of 40 μg/ml of the supernatant30 .

Chromatin Immunoprecipitation (ChIP) protocol was performed as described by [82 ]. Briefly, the wild-type, 35S::XAL1/xal1-2, and 35S::XAL1-GFP/xal1-2 lines were grown on MS 0.2x plates for ten days and the material (1.0 g) was fixed. Chromatin was solubilized using a sonicator (Fisher Scientific; power 20%) in 20 cycles of pulses 15 s each. Immunoprecipitation was conducted overnight using an anti-GFP rabbit IgG fraction (A11122, Invitrogen; 1:5000 dilution) with A/G agarose beads (Thermo Fisher). Template ChIP DNA was amplified using primer pairs designed within or in the flanking regions of CArG boxes found primarily in the 5′ intergenic region of the FD, SOC1, LFY, and AP1 genes (Table S2). ACTIN 2 was used as a constitutive reference gene. ChIP experiments were performed in triplicates, and the data was normalized against the 35S::XAL1/xal1 control sample.