Bradford protein quantification kit

Protein was isolated from hepatic tissue using lysis buffer enrich with phosphatase and protease inhibitors (Keygen biotech, Nanjing, China) and quantitated using Bradford protein quantification kit (Yeasen, shanghai, China). Equal amounts of protein were loaded and separated on sodium dodecyl sulphate polyacrylamide gels and then incubated with specific antibodies. The following primary antibodies were used: GLK (bs-1796R), PEPCK1 (bs-4972R), PFK1 (bs-3982R), CPT1A (bs-2047R) from Bioss Inc. (Beijing, China); G6P (sc-398,155) from Sant Cruz biotecnology (CA, USA); HMGCR (BM4908) from Boster biological technology (Wuhan, China). Images were captured with G: BOX chemiXR5 imaging System (Syngene, Cambridge, UK) and analyzed with Gel-Pro32 software.

PLGA copolymer (75:25, Mol. Wt. = 60 kDa) was purchased from the Daigang Biological Co. Ltd. (Jinan, China). Potassium bromide (KBr), absolute ethanol (99.8% purity), tetrahydrofuran (THF), and dichloromethane (DCM, 99.8% purity) were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lysozyme ~7000 U/mg was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals, solvents, and reagents were of analytical purity and used without any further purification. Cell lysate, Roswell Park Memorial Institute 1640 (RPMI-1640), α- minimum essential medium (α-MEM), albumin bovine V, and Fetal bovine serum (FBS) were acquired from Gibco (USA). Cell Counting Kit-8 (CCK-8) was purchased from KeyGEN BioTECH, Jiangsu, China. Bradford Protein Quantification Kit was purchased from YEASEN, Shanghai, China.

A volume (1 ml) of urea pyrolysis solution [20 mM 2-hydroxyethyl (HEPES), 9 M urea, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 mM β-glycerophosphate, pH 8.0] was added to each tissue sample (100 mg) for an ice-bath ultrasonic treatment (100 W, 10 s, interval 10 s, 10 times). The solution was centrifuged (18,000 × g, 30 min, 4°C). The supernatant was the protein extraction, and its protein content was determined with a Bradford Protein Quantification Kit (YEASEN, Cat# 20202ES76).

Protein extraction from egg yolk was performed according to Mann and Mann, (2008) procedure [5 (link)]. Briefly, 0.2 g of egg yolk was added in 1 mL ice-cold lysis buffer (5 μL of phosphatase inhibitor, 1 μL of protease inhibitor, 10 μL of phenylmethanesulfonyl fluoride) and then homogenized at 4 °C for 3 min. The samples were centrifuged at 12,000× g for 10 min at 4 °C and whole yolk protein extract was collected. For desalination of yolk protein extract, a 100 mL extracted protein was mix with 400 mL of ice-cold acetone and kept overnight at −20 °C, then the sample was centrifuged at 12,000× g for 10 min and the pellet was air-dried for 15 min in a draft cupboard (FGG1500, Kebei, Wuhan, China) and then through rehydration buffer-1 (8-mol urea, 4% CHAPS, 65-mmol dl-dithiothreitol, Bio-Lyte 0.2% (w/v), and bromophenol blue 0.001%) resolubilized. The samples were homogenized for 10 min and then centrifuged at 1000× g for 10 min at 4 °C. The supernatant was collected and stored at −20 °C. Bradford protein quantification kit was used for the quantification of protein samples (Yeasen, Shanghai, China).

The specific activity of aMOx and mutants was determined by evaluating the consumption of NADPH (Liu et al., 2021 (link)). The Bradford (1976) (link) method was used to determine the protein concentration using a Bradford protein quantification kit (YEASEN Biotechnology Co., Ltd., Shanghai, China). The reaction conditions were as follows: 0.1 μM purified enzyme, 0.1 mM styrene, and 0.2 mM NADPH in 25 mM Tris-HCl, at pH 8.0. The reaction volume was 200 μl. The reaction system was shaken for 30 min at 25°C. Then, a Multiskan Sky microplate spectrophotometer (Thermo Scientific, Waltham, MA, United States) was used to detect the change of absorbance value at 340 nm within 30 min.

GUS measurements were carried out as described previously (Haq et al., 2022 ). Promoter fragments of OsSWEET11a and Xa27 were cloned into the binary GUS reporter construct pCAMBIA1381 using primers listed in Supplemental Table 3. The binary vector pHB was used to clone and express TALEs PthXo1 and Tal6b in N. benthamiana. The effector and reporter constructs (Supplemental Table 1) were transformed into Agrobacterium strain EHA105 by the freeze–thaw method and then co-expressed (OD₆₀₀ = 1.0 for each strain) in 5- to 7-week-old N. benthamiana leaves via Agrobacterium-mediated transformation. Three leaf discs (1 cm diameter) were collected 48 h post inoculation, and GUS activity was measured using 4-methylumbelliferyl-β-glucuronide. Proteins were quantified using a Bradford Protein Quantification Kit (Yeasen, Shanghai).