Bicinchoninic acid bca assay kit

The genomic DNA of S. aureus isolated from Ningxia Province was used as a template to amplify blaZ, the gene encoding β-lactamase, by PCR, and the amplification products were sequenced for comparison. Subsequently, BamHI and Xhol I enzymatic (New England Biolabs [NEB]) cleavage sites and protective bases were introduced at both ends of the blaZ gene. The upstream primer was 5′-CGCGGATCCAAAGAGTTAAATGATTTA-3′, and the downstream primer was 5′-CCGCTCGAGTCAAAATTCCTTCTATACACT-3′ (restriction sites are underlined). The pET28a-blaZ recombinant plasmid was constructed and verified by double digestion; subsequently, gel recovery, ligation, and transformation were carried out. The positive clones were screened in LB agar (kanamycin resistant) and identified by sequencing, followed by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma-Aldrich) to a final concentration of 1 mM in the bacterial culture fluid. Furthermore, Escherichia coli BL21(DE3) organisms were collected at 12,000 rpm after 6 h of induction at 16°C, and the expression of the target protein was observed by SDS-PAGE. The BlaZ protein was purified by Ni ion affinity chromatography column. In addition, high concentrations of imidazole were removed by centrifugal filter devices (Millipore; 3 kDa), and the concentration was determined with a bicinchoninic acid (BCA) assay kit (Sigma-Aldrich).

[³H]Aβ_1–40 (20 Ci/mmol) and [³H]desmosterol (10 Ci/mmol) were purchased from Hartmann Analytic (Braunschweig, Germany), [¹⁴C]sucrose (435 mCi/mmol), liquid scintillation cocktails Ultima Gold XR® and Solvable® were purchased from PerkinElmer Life Sciences (Courtaboeuf, France). L-thyroxine (T₄), probucol, desmosterol, Triton X-100, human serum, and bicinchoninic acid (BCA) assay kits were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). All the other chemicals were commercial products of reagent grade.

Human angiotensin II (Ang II), GS-4997 (an ASK1 inhibitor), and bicinchoninic acid (BCA) assay kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against the following proteins were used: α-smooth muscle actin (α-SMA), apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, collagen type I (Col I), and Col III (Cell Signaling Technology Inc., Beverly, MA, USA); GRP78, PERK, p-PERK, and CHOP (Abcam, Cambridge, MA, USA); and CD63, TSG, and GAPDH (Santa Cruz Biotechnology Inc., Dallas TX, USA). A fluorescein isothiocyanate- (FITC-) conjugated horseradish peroxidase- (HRP-) conjugated secondary antibody and an enhanced chemiluminescence kit were obtained from the Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). A reactive oxygen species (ROS) assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA); and Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA, USA).

Five flies were homogenized in 200 µL of PBS and centrifuged (3 min, 4°C, 8000 RPM) for glycogen measurement. For lactate and glucose measurement, hemolymph was isolated from 25 adult males by centrifugation (14,000 RPM, 5 min) through a silicagel filter into 50 µL PBS. Half of all samples were used for the quantification of proteins. Samples for glucose, glycogen and lactate measurement were denatured at 75°C for 10 min, whereas samples for protein quantification were stored in −80°C. Glucose was measured using a Glucose (GO) Assay (GAGO-20) Kit (Sigma) according to the manufacturer’s protocol. Colorimetric reaction was measured at 540 nm. For glycogen quantification, sample was mixed with amyloglucosidase (Sigma) and incubated at 37°C for 30 min. A Bicinchoninic Acid Assay (BCA) Kit (Sigma) was used for protein quantification according to the supplier's protocol and the absorbance was measured at 595 nm. A Lactate Assay Kit (Sigma) was used for lactate concentration quantification according to the manufacturer's protocol. The absorbance was measured at 570 nm. Samples for metabolite concentration were collected from six independent experiments. Measured data were compared in Graphpad Prism using Tukey's multiple comparisons test. Sidak's multiple comparison correction was performed.

Edwardsiella bacterins, whole-cell lysate (WCL) was prepared from E. tarda strain ED-BDU1. For vaccine preparation and immunization, ED-BDU1 cells with an OD of 1.0 were harvested by centrifugation at 4,000 g for 15 min and washed three times with saline solution. Washed cells were suspended in sterile saline solution as a live E. tarda vaccine or then incubated at 100°C to produce the inactivated vaccines. Plate counting assay was performed to examine bacterial sterility in the inactivated vaccines. HflC, HflK, and YhcI recombinant proteins were over-expressed and purified using the E. coli expression system as described above. The recombinant proteins were dialyzed, and the protein concentration was determined using a Bicinchoninic acid assay (BCA) kit (Sigma, St. Louis, MO). Then 100 µg of protein in Alhydrogel (Sigma, St. Louis, MO) was used as a vaccine formulation for the first booster on day 0. The second booster of 100 µg protein was administered by subcutaneous injections on the 21^st day.