Protein assay cbb solution

To generate recombinant CARSs, open-reading frames of these genes were transferred into AG1 (Agilent Technologies, Santa Clara, CA) competent cells. Recombinant EcCARS, mouse CARS1, and human CARS2 proteins were purified by using the following standard procedure. Briefly, these proteins were produced in AG1, and they were purified by using nickel nitrilotriacetic acid agarose; resultant purified proteins were extensively dialyzed against phosphate buffer and stored at −80 °C until use. Protein concentration was determined by using the Protein Assay CBB Solution (Nacalai Tesque, Kyoto, Japan), and protein purity was confirmed via SDS-PAGE.

Water-soluble progesterone (PG), anesthetic MS-222 and apocynin were obtained from Sigma (St. Louis, MO, USA). Collagenase (280 U/mg) and catalase (11,000 U/mg) were purchased from Wako (Osaka, Japan), hCG was from Teikoku Zoki (Tokyo, Japan). Fluorogenic caspase-3 substrate IV was purchased from Calbiochem (La Jolla, CA, USA). Hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA, USA). Polyclonal anti-MAPK and anti-pMAPK antibodies were from Cell Signaling (Beverly, MA, USA), biotinylated anti-rabbit IgG was from Vector Laboratories (Burlingame, CA, USA). The Streptavidin Biotin Complex Peroxidase Kit, protein assay CBB solution, SOD (5000 U/mg) and BHA were from Nacalai Tesque (Kyoto, Japan). 2′,7′-dichlorofluorescein diacetate (DCFDA) Cellular ROS Detection Assay Kit was obtained from Abcam (Cambridge, UK), EUK 134 and MITO-TEMPO were ordered from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA). Other chemicals were obtained from Wako and Nacalai Tesque.

Anesthetic MS-222, water-soluble progesterone, and ATP Bioluminescence Assay Kit CLS II were purchased from Sigma (St. Louis, MO). Collagenase (280 U/mg) was obtained from Wako (Osaka, Japan) and human chorionic gonadotropin was from Teikoku Zoki (Tokyo Japan). Hydrogen peroxide, Sudan Black B (SBB), and protein assay CBB solution were from Nacalai Tesque (Kyoto, Japan). Hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA). Other chemicals were obtained from Wako and Nacalai Tesque. Slide glasses and cover slips for microscopy were purchased from Matsunami Glass (Osaka, Japan).

Anesthetic MS-222 and water-soluble PG were purchased from Sigma (St. Louis, MO, USA). Collagenase (280 U/mg) was obtained from Wako (Osaka, Japan) and hCG was from Teikoku Zoki (Tokyo Japan). Polyclonal anti-MAPK and anti-pMAPK antibodies were from Cell Signaling (Beverly, MA), biotinylated anti-rabbit IgG was from Vector Laboratories (Burlingame, CA, USA), and the Streptavidin Biotin Complex Peroxidase Kit was from Nacalai Tesque (Kyoto, Japan). Polyvinulidene difluoride (PVDF) membranes Immobilon were purchased from Merck Millipore (Carrigtwohill, Ireland). Protein assay CBB solution was from Nacalai Tesque (Kyoto, Japan). Specific MEK inhibitor U0126 was from Promega (Madison, WI) and MMP inhibitor GM6001 was from Calbiochem (Darmstadt, Germany). Other chemicals were obtained from Wako and Nacalai Tesque.

E. coli BL21(DE3) cells transformed with the plasmid encoding TfCut2 and its mutants (except G62A/F209A) were cultured in LB medium at 37 °C to OD₆₀₀ = 0.4. TfCut2 expression was induced by the addition 0.1 mM isopropyl-D-thiogalactopyranoside at 37 °C for 4 h. The cells were harvested by centrifugation (4 °C, 10,000 × g, 10 min), the cell pellets were resuspended in 20 mM Tris-HCl (pH 8.0), then lysed by sonication on ice. Cell debris was removed by centrifugation (4 °C, 10,000 × g, 10 min) and the supernatant was applied to a Ni-NTA agarose column (Qiagen). The column was washed with wash buffer (20 mM Tris-HCl, 40 mM imidazole, pH 8.0) and TfCut2 was eluted using elution buffer (20 mM Tris-HCl, 100 mM imidazole, pH 8.0). The fraction containing TfCut2 was dialyzed against 20 mM Tris-HCl (pH 8.0) at 4 °C and the concentration of TfCut2 was measured using protein assay CBB solution (Nacalai Tesque).

For each A. oryzae strain, 50 mL of PD medium was prepared with and without acetic acid (final concentration 5.3 or 7.9 g/L). The pH was adjusted to 6 using 2 M HCl (without acetic acid) or NaOH (with acetic acid). Spore suspensions of A. oryzae strains RIB40 and AOK27L were inoculated in the medium (final concentration 1 × 10⁴ spores/mL) and incubated for 72 h at 30 °C. The supernatants were filtered through a 0.22 μm Durapore membrane (Millipore). Quantification of the protein amount was performed using Protein Assay CBB Solution (Nacalai Tesque), as described before.

The cells were lysed with RIPA buffer (Nacalai Tesque) for 15 min on ice and centrifuged at 10 000×g for 10 min at 4 °C to remove insoluble material. Protein concentration was determined by a Protein Assay CBB Solution (Nacalai Tesque). The cell lysates (24 μg) were separated by electrophoresis on 5–20% Extra PAGE One Precast Gel (Nacalai Tesque) at 200 V for 45 min. The protein was transferred to PVDF membrane by a Trans-Blot Turbo Transfer System (BIO-RAD). The membranes were incubated in 5% skim milk for 1 h at room temperature to prevent nonspecific binding. Then, the membranes were incubated in primary antibodies against HER2 (1 : 5000, Santa Cruz Biotechnology), EGFR (1 : 100, Santa Cruz Biotechnology), VEGFR-2 (1 : 200, Santa Cruz Biotechnology), PD-L1 (1 : 500, CUSABIO) or beta-actin (1 : 200, Santa Cruz Biotechnology) for overnight at 4 °C. After washing with TBST, the membranes were incubated with a 1 : 2000 dilution of secondary antibody (goat anti-mouse IgG, HRP conjugate, Millipore) for 1 h at room temperature. After washing with TBST, bands were visualized by treating the membranes with ImmunoStar LD (FUJIFILM) according to manufacturer's instructions and detecting the bioluminescence with LAS-4000 (GE).