Coomassie brilliant blue

EMSA was performed essentially as described^{34 (link)}. Recombinant TrhO (0, 10, 20, 40, or 80 pmol) and in vitro transcribed tRNA^Ala1 or tRNA^Leu3 (20 pmol each) were incubated at 37 °C for 1 h in 10 µl of reaction mixture [50 mM HEPES-KOH (pH 7.5), 5 mM Mg(OAc)₂, 100 mM KCl, 1 mM spermine, 1 mM DTT]. The mixtures were electrophoresed in 6% native polyacrylamide gel with running buffer [50 mM HEPES-KOH (pH 7.5), 5 mM Mg(OAc)₂, and 1 mM DTT] in cold room. The gel was first stained with SYBR Safe (Thermo Fisher Scientific) to detect tRNA and then with Coomassie brilliant blue (Nacalai Tesque) to detect protein.

SDS–PAGE was carried out using a 15% polyacrylamide gel. The fusion proteins were observed by staining with Coomassie Brilliant Blue (Nacalai Inc., Kyoto, Japan) and Periodic acid-Schiff (PAS). Western blotting was carried out with primary antibodies of anti-human IFNα2b and anti-HSA. It was then further reacted with secondary antibodies of anti-mouse IgG (IFNα2b) and anti-goat IgG (HSA) conjugated to HRP. The specific protein bands were visualized using a chemiluminescence kit. ESI-TOFMS analysis for Man-HSA(D494N)-IFNα2b was performed as previously reported (Miyamura et al., 2016 (link)).

Approximately 35 h after inoculating conidia of OU14 and RACE1 isolates on the primary leaves bombarded with TIGS constructs, GUS staining was performed as described by Yaeno et al. [34 (link)] and Bgh was stained with Coomassie brilliant blue solution (0.3% w/v Coomassie brilliant blue R-250 [Nacalai Tesque, Kyoto, Japan], 7.5% trichloroacetic acid, and 30% methanol) and washed three times with water. The penetration rate was calculated by counting the number of conidia developing haustoria in GUS-expressing cells per total number of interactions between conidium and a GUS-expressing cell.

The PspA gene was amplified by polymerase chain reaction (PCR) and cloned into pET16b plasmid (Novagen, Darmstadt, Germany), as previously described, to yield pET16b-PspA plasmid [17 (link)]. To obtain PspA recombinant proteins, the plasmids were transformed into E. coli strain BL21 (DE3) (Novagen). Protein production was induced by adding isopropyl-β-D-thiogalactopyranoside (Nacalai Tesque, Kyoto, Japan). The culture pellets were sonicated for 1 min three times in buffer A (10 mM Tris-HCl [pH 8.0], 400 mM NaCl, 5 mM MgCl₂, 0.1 mM PMSF, 1 mM 2-mercaptethanol, and 10% glycerol). After centrifugation of the mixture at 4 °C and 17,800× g for 15 min, the supernatants were filtered through a 0.45 µm Millex-HV filter unit (Merck Millipore, Burlington, MA, USA) and loaded into HiTrap HP columns (GE Healthcare, Pittsburgh, PA, USA). PspA was eluted with buffer A containing 100 to 500 mM imidazole. The eluted protein was loaded into a PD-10 column (GE Healthcare) for exchange with PBS (Nacalai Tesque). The concentration of purified protein was measured by using a BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). The purity of the eluted protein was confirmed in a NuPAGE electrophoresis system (Life Technologies, Carlsbad, CA, USA) followed by staining with Coomassie brilliant blue (Nacalai Tesque).

ɑSyn fibrils (0.5 mg/mL) in the fibrillation buffer were digested using proteinase K (03115887001, Sigma) (1 µg/mL) at 37 °C and agitation at 400 rpm for different time intervals. To stop the reaction, the samples were incubated at 95 °C for 5 min, mixed with loading buffer (1610747, Bio-Rad) (50 mM Tris–HCl, pH 6.8, 4% SDS, 2% β-mercaptoethanol, 12% glycerol, and 0.01% bromophenol blue) and incubated at 95 °C for an additional 10 min. The digestion patterns were analyzed using SDS-polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue (11642-31, Nacalai Tesque, Kyoto, Japan) staining. The first five digestion products, B1–B5, were used for analysis. The proteinase K resistance (PKR) score was established as the band intensity ratio between bands B2 and B1 (B2/B1).

Briefly, 1 × 10⁷ MOLT-3 cells were lysed in buffer (1% Triton X-100, 10 mM Tris, 150 mM NaCl, pH 7.6). A four-step procedure for the purification of the PTE-associated proteins was performed. In the first step, the lysate was added to Sepharose 6B beads and incubated with gentle rocking overnight at 4°C. The samples were then centrifuged and the supernatant was collected. In the second step, the supernatant was added to the GTE Sepharose 6B beads and incubated with gentle rocking overnight at 4°C, and the supernatant was collected by centrifugation. In the third step, the second-step supernatant was added to BTE Sepharose 6B beads, which were then incubated overnight at 4°C, and the supernatant was isolated by centrifugation. In the final step, the third-step supernatant was added to PTE Sepharose 6B beads and the supernatant was incubated overnight at 4°C. The PTE Sepharose 6B beads were then washed five times with lysis buffer. The proteins that were bound to the beads were analyzed on SDS-PAGE. Cell lysates without any treatment served as the controls. The gels were stained with Coomassie brilliant blue (Nacalai, Kyoto, Japan).