Bradford protein assay kit

Western blotting was performed as previously described^{30 (link)}. Total protein from cells (10 μg) and exosomes (10 μg) was fractionated using an electrophoretic gradient across Mini-PROTEAN tris-glycine extended gels (BIO-RAD, Richmond, CA, USA). Measurement of protein concentration was performed using a Bradford Protein Assay Kit (Takara Bio), according to the manufacturer’s instructions. The gels were then transferred onto Immun-Blot PVDF membranes (BIO-RAD) under wet electrophoretic conditions. The blotted protein was blocked for 1 hr at room temperature with Odyssey blocking buffer in PBS (LI-COR, Lincoln, NE, USA) and was followed by incubation overnight at 4 °C with the following primary antibodies: 1:1000 anti-CD63 mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA); 1:1000 anti-cytochrome-c mouse monoclonal antibody (BD Biosciences); 1:1000 anti-calnexin rabbit monoclonal antibody (Abcam) and 1:1000 hFAB Rhodamine (#12004168 - Bio-Rad). Primary antibodies were detected by IRDye 800CW anti-rabbit IgG and IRDye 680RD anti-mouse IgG secondary antibodies (LI-COR) and were incubated with the protein-blotted membrane for 1 hr at room temperature. Fluorescence was then detected on the Odyssey imaging system (LI-COR).

The strain GS115 (pro/rDNA-mtg) of higher enzyme activity was cultured in 100 mL BMGY medium for 24 h and controlled at 28 °C. Then the cell was harvested by centrifugation and inoculated into 200 mL BMMY medium at different initial pH (5.0, 6.0, 7.0 and 8.0) for 72 h and maintained at 25 °C. 1 M Na₂HPO₄•12H₂O (pH = 8.85) and 1 M KH₂PO₄ (pH = 4.01) were mixed in a certain proportion to form phosphate buffer with buffer range pH 4–9 and used to adjust the initial pH of BMMY medium. The cell density (OD600) and the activity of MTG were detected as described below. Besides, the cell density and the activity of MTG were measured once every 12 h at different inducted temperatures (i.e. 20, 25 and 30 °C).
The activity of MTG was measured as Yurimoto described [21 (link)]. The MTG enzyme activity was defined as follows: catalytic substrate (N-CBZ-Gln-Gly) to generate 1 μmol glutamine hydroxamate (hydroxamic acid) at 37 °C as one enzyme activity unit. Protein concentrations were determined by using a Bradford Protein Assay Kit (TaKaRa) and the standard protein was bovine serum protein (Thermo) [21 (link)].

Human adipose tissues were obtained from four female donors aged no less than 18 years-old, undergoing cosmetic liposuction from the thigh at the related facility after obtaining written informed consent (Figure 5). Liposuction from the thigh was not indicated to the patients with BMI less than 17.5 or greater than 35 kg/m², previous thigh surgery, comorbidities, or regular medicine that prohibit the surgery or anesthesia. The mean age and body mass index (BMI) of donors were 31.7 (range: 24–37) years and 20.4 (range: 17.9–22.5) kg/m², respectively. The lipoaspirates were poured into disposable sterilized 50 mL syringes with a filter piston (Medikan Corp., Seoul, Korea) and centrifuged at 1200× g for 3 min using a Lipokit^® device (Medikan) [27 (link)]. After centrifugation, samples were separated into three fractions: oil (onto the piston), adipose (middle: concentrated adipose tissue), and fluid (bottom: containing the blood cell component). The concentrated adipose tissue was collected and mixed with an equal volume of PBS. AE was obtained by centrifuging at 2600× g for 10 min (Model 2800, Kubota Co., Tokyo, Japan), then filtering the aqueous portion and removing cellular and tissue debris. The protein concentration of AE was measured using a Bradford Protein Assay Kit (TaKaRa Bio Inc., Shiga, Japan). All the procedures were performed under sterile conditions.

For the in vitro cleavage assay, the TnpB–ωRNA complexes (wild-type or mutants) were purified in a similar manner to that for the complex prepared for the cryo-EM analysis. Protein concentrations were measured using a Bradford Protein Assay Kit (TAKARA). The DNA cleavage activity of TnpB was measured by in vitro DNA cleavage assays. The TnpB–ωRNA complex (2 μl, final concentration 250 nM) was mixed with the 3-kb linearized plasmid target containing the 16-nt target sequence and the TTGAT TAM (8 μl, 100 ng) (Supplementary Table 1), and incubated at 37 or 50 °C for 30 min in 10 μl reaction buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 5% glycerol). The reaction was stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (Shimadzu). In vitro cleavage experiments were performed at least three times.

The Alexa Fluor™ 488 protein labeling kit (Thermo Fisher Scientific, Waltham, MA, USA) was applied to fluorescently label the CD81 protein. According to the user guide, 84 mg of sodium bicarbonate was first dissolved in 1 mL of Milli-Q to prepare 1 M sodium bicarbonate solution. The CD81 solution was then prepared at 0.2 mg/mL by dissolving 100 µg of CD81 in 500 µL of 0.1 M sodium bicarbonate buffer (pH 8.3). The obtained CD81 solution was then applied to a vial containing Alexa Fluor™ 488 fluorescent dye with a succinimidyl ester group, followed by incubation on a magnetic stirrer at room temperature in the dark for 3 h. The excess dye was purified using the Slide-A-Lyzer™ dialysis cassette (3.5K MWCO, 0.5 mL, Thermo Fisher Scientific, USA) and 100 mM Tris–HCl buffer. The concentration of the labeled CD81 protein solution was finally quantified using a Bradford protein assay kit (Takara Bio Inc., Shiga, Japan).

Western blotting was carried out as described previously^{55 (link)}. In brief, cells were washed with cold PBS 3 times to completely remove nanoparticles, and then harvested and lysed in RIPA buffer (9806, CST) containing protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1% phosphatase inhibitor (P5726, Sigma-Aldrich). The lysates were centrifuged at 14000 rpm for 30 min at 4 °C. The protein concentration was measured using a Bradford protein assay kit (T9310A, Takara, Shiga, Japan). Protein samples were then mixed with a 2 × Laemmli sample buffer (161–0737, Bio-Rad, CA, USA) and heated at 95 °C for 5 min. Equal amounts of protein were then applied to 5–20% SuperSep® Ace precast gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and transferred to Immun-Blot PVDF membranes (1620174, Bio-rad). The membranes were blocked with 5% skim milk buffer for 1 h at room temperature, and then incubated overnight at 4 °C with the following ab: anti-ACTB (1:5000), anti-MAP1LC3B (1:1000), and anti-p62/SQSTM1 (1:2000). Lastly, they were incubated for 1 h with HRP-conjugated anti-mouse or anti-rabbit ab and visualized with an enhanced chemiluminescence detection system (32106, PIERCE, Rockford, IL, USA).