Bradford protein assay kit

200 mL of LB medium and 40 mg/L kanamycin were added in 1 L shake flask. The transformed E. coli BL21 (DE3) harboring fusion enzyme or free enzyme plasmids were cultured at 37 °C, 200 rpm in 1 L shaking flask. The expression of recombinant proteins was induced by the addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the optical density (OD600) was reached at 0.6–0.8 and the E. coli was grown at 25 °C for 16 h. Cell was collected by centrifugation and then resuspended with PBS buffer. Cell was disrupted by ultrasonication and removed by centrifugation at 10,000 rpm, 4 °C for 20 min. Obtained crude cell extract was then added to His-Trap column (His-Trap HP 5 mL, GE Healthcare Corp., Piscataway, NJ, USA) which pre-equilibrated with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4). The binding buffer was equilibrated the column and eluting buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) eluted at a gradient concentration. Target proteins were desalted and concentrated by Macrosep Advance Centrifugal Devices (cut-off 10 kDa, Pall, East Hills, NY, USA). The purity of the obtained enzymes was tested by 10% (w/v) SDS-PAGE. All the protein concentrations were determined using a modified Bradford Protein Assay Kit (Sangon Biotech Co. Ltd) with bovine serum albumin as standard.

The enzyme activities of mitochondrial isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (KGDH), hexokinase (HK), pyruvate kinase (PK), and phosphofructokinase (PFK) were analyzed using detection kit (Solarbio, Beijing, China), respectively, according to the manufacturers’ protocols. All values were normalized to the total protein levels. The protein content was quantified by the Bradford Protein Assay Kit (Sangon, Shanghai, China). All these enzymatic activities in the control group were normalized to 1.0.

To detect the gene expression profile in intestine, qRT-PCR analysis was performed. Total RNAs were extracted using the TRIzol. The first-strand cDNA was synthesized using a ReverTra Ace qPCR RT Kit (FSQ-101, Toyobo, Osaka, Japan) according to the manufacturer’s instruction, and was used as the template for the quantitative real-time PCR (qPCR). qPCR was performed by using the iQ SYBR Green Supermix (170–8882, Bio-Rad, Hercules, CA, USA) and the CFX96 Real-Time System (Bio-Rad). The PCR program was set as: 95°C for 10 min; 40 cycles at 95°C for 15 s, 60°C for 50 s, and plate reading at 72°C for 2 s; and then a melting period from 65 to 95°C. The data were processed using the 2^−ΔΔCT method [41 (link)].
For protein level analysis, total intestine proteins were extracted by homogenizing the tissue using Radioimmunoprecipitation assay (RIPA) Lysis Buffer (P0013B, Beyotime, Wuhan, China). The tissue homogenate was centrifuged at 12,000 × g for 15 min. The resultant supernatant was collected to determine the protein concentration using a Bradford protein assay kit (C503031, Sangon Biotech, Shanghai, China), mixed with Protein Loading Dye (P0015L, Beyotime) and boiled at 100°C for 10 min for subsequent electrophoresis and western blotting analysis. At least five shrimp were used for each sample.

To determine the distribution of FoxO and Relish, nuclear proteins were extracted using a Nuclear Protein Extraction Kit (R0050, Solarbio), according to the manufacturer’s instructions. Generally, shrimp intestines were homogenized with cytoplasmic protein extraction reagent containing 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was shaken for 20 s and then placed on ice for 3 min. After five times of alternative treatment, the homogenate was centrifuged at 13,000 × g for 20 min at 4°C. After three washes using PBS, the sediment was resuspended with nucleoprotein extraction reagent containing 1 mM PMSF, and processed as above. After centrifugation at 13,000 × g for 20 min at 4°C, the supernatant was collected as the nuclear proteins. The protein concentration was determined using a Bradford protein assay kit (C503031, Sangon Biotech). At least five shrimp were used for each sample.

The antibody titer of PAbs were determined by indirect ELISA according to a previously described method [34 ]. Briefly, the concentration of the PAbs purified by CAAS and the specific PAbs were determined by Bradford Protein Assay Kit (Sangon, C503031, China) and then diluted to 1 mg/mL. The wells of a 96-well microtiter plate was coated with 100 μL of 5 μg/mL recombinant PCV2 dCAP protein and incubated overnight at 4 °C. The unbound protein was discarded and the plate was washed three times with PBST. After blocking for 1 h, the plate was also washed three times with PBST and incubated with the PAbs diluted with PBST at 1:1000 for 2 h at 37 °C. And with three times washing, the plate was incubated with HRP-Goat anti-Rabbit polyclonal antibody (Affinity, S0001, China) diluted with PBST at 1:3000 for 2 h at 37 °C. Finally, the plate was washed for three times and incubated with 200 μL TMB (beyotime, P0209, China) for 10 to 30 min at 37 °C and added with 50 μL of stopping solution. The absorbance at 450 nm was measured with a microtiter plate reader.

The total cell protein was extracted on ice using lysis buffer (50 mM Tris-HCl, pH = 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1 mM EDTA, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 0.1 mM Na3VO4, 0.1 mM NaF, and cocktail protein inhibitor). The protein concentrations were determined with a Bradford bioassay using a Bradford protein assay kit (Sangon). Protein (20 ug) samples were electrophoresed in 4% stacking and 10%/15% resolving SDS-PAGE gels, and the fractionated proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Amersham Bioscience). Blots were blocked with 5% non-fat milk at room temperature for 1 h and were incubated with primary antibody overnight at 4°C. After being washed with TBST, the blots were incubated with secondary antibody for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence substrate (Tanon) and then quantified using a Tanon Chemiluminescent Imaging System.