Immobilon p pvdf transfer membrane

Ten percent BH, (10% w/v) were prepared on ice in PBS Sarkosyl 2% and aliquots digested with 20U/ml PK (corresponding to 400 µg/ml when PK specific activity is 50U/mg) (Roche Diagnostics) for 1 h at 37 °C. 1 ml of PMCA-untreated vCJD-urine was centrifuged at high speed (100,000 × g for 1 hour at 4 °C) and PK digestion (5U/ml, corresponding to 100 µg/ml when PK specific activity is 50U/mg), was performed for 1 h at 37 °C on the pellet resuspended in 10 µl of PBS Sarkosyl 2%. PK digested. The PK digested samples were diluted in sample buffer (final concentration: 3% SDS, 4% β-mercaptoethanol, 10% glycerol, 2 mM EDTA, 62.5 mM Tris, pH 6.8) and boiled for 8 min before loading. Protein samples were separated with Tris-Glycine SDS-PAGE in 15% Criterion Tris-HCl polyacrylamide precast gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to Immobilon-P PVDF transfer membrane (EMD-Millipore, Billerica, MA, USA) for 2 h at 60 V, blocked with 5% nonfat dry milk in 0.1% Tween 20-Tris-buffered saline, pH 7.5, and probed with the monoclonal anti-PrP antibody 3F4^{27 (link)}. The immunoreactivity was visualized by enhanced chemiluminescence (Pierce ECL 2, Fisher Scientific, Hampton, NH, USA) on Kodak BioMax Light films (Eastman Kodak Co., Rochester, NY, USA).

The following reagents were used: anti-HERC2 monoclonal (BD Biosciences #612366); anti-HERC2 polyclonal (Bvg1 antibodies against residues 4785-4834, and Bvg9 antibodies against residues 1-199) [15 (link)]; anti-p21 (C-19), anti-p53 (FL-393), anti-NEURL4 (E-20) (Santa Cruz Biotechnology, Inc.); anti-p53 Ab-5 (DO-7) (Neo Markers); anti-GST monoclonal (GenScript); anti-Ran [34 (link)]; anti-α-tubulin (Ab-1) (Calbiochem); anti-USP33 (Proteintech); anti-Flag M2 (Sigma); anti-GFP and anti-c-myc (clone 9E10) (Roche); anti-MDM2 (2A10) (Abcam); Z-Leu-Leu-Leu-al (MG132) (Sigma-Aldrich); horseradish peroxidase-conjugated secondary antibodies; lipofectamine LTX (Invitrogen); cycloheximide (Applichem); protein A-Sepharose and glutathione-Sepharose (GE Healthcare); GFP-Trap_A (ChromoTek); Immobilon-P PVDF transfer membrane (Millipore Corporation); luciferase assay system (Promega); luminescent β-galactosidase detection Kit II (Clontech Laboratories).

Cell-free synthesized proteins or cell culture supernatants containing inactivated MERS-CoV were mixed with an equal volume of 2X SDS sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0.01% bromophenol blue] and heated at 100°C for 5 min. After separation by 12.5% or 15% SDS-PAGE using Hi-QRAS Gel N (Kanto Chemical, Tokyo, Japan), the proteins were electrotransferred onto an Immobilon-P PVDF Transfer Membrane (Millipore, Bedford, MA, USA) as described previously (Nishi et al., 2014 (link)). The membrane was blocked in Tris-buffered saline (TBS) containing 2% (w/v) skim milk for 30 min, and then incubated for 1 h with anti-MERS-NP mAbs or anti-His polyclonal antibody (GTX115045; GeneTex, Irvine, CA, USA) in TBS containing 0.1% (v/v) Tween 20 (TBS-T; 1:1000 dilution) and 0.4% (w/v) skim milk. After three washes with TBS-T, the membrane was incubated for 60 min in PBS containing HRP-conjugated goat-anti mouse or rabbit IgG antibody (1:10000 dilution; GE Healthcare). After an additional three washes in TBS-T, proteins were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Rockford, IL, USA) or Immobilon (Millipore, Bedford, MA, USA) on a Lumi-Imager F1 (Roche Diagnostics, Basel, Switzerland).

To determine the expression effects of NPF protein levels, the coding sequences of npf were inserted into the linearized pET28a (+) expression vector. The construct containing npf genes was transformed into E. coli BL21 cells (Tiangen, Beijing, China) and induced with a final concentration of 1 mM IPTG for expression. The expression product was transferred to ABclonal Company to synthesize rabbit anti-NPF (ABclonal Technology, Wuhan, China). The midgut was homogenized and lysed in RIPA lysis buffer (CWBio, Beijing, China) supplemented with protease inhibitor cocktail. Protein contents were determined with a BCA Protein Assay Kit (Beijing BioDee Biotechnology, Beijing, China). Next, 20 μg of protein was separated with 15% SDS-PAGE and transferred to an Immobilon-P PVDF transfer membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in TBST at room temperature for 2 h, the membranes were incubated overnight at 4 °C with primary antibody specific to rabbit anti-NPF (1:500) and mouse anti-β-tubulin (1:2000). The membranes were washed in TBST and then incubated with HRP goat anti-rabbit IgG (H+L) and HRP goat anti-mouse IgG (H+L) (ABclonal Technology, Wuhan, China) (1:2000) at room temperature for 2 h. The signals were captured using the Azure Biosystems C600 (AZURE Biosystems, Dublin, CA, USA) and quantified using ImageJ (NIH, Bethesda, MA, USA).

After washing cells twice with PBS, whole cell lysates were prepared using M-PER Mammalian Protein extraction Reagent (Thermo Scientific, Rockford, IL). Protein concentration was determined using the bicinchoninic acid (BCA) assay. Equal amount of protein was separated by electrophoresis on SDS-polyacrylamide gels and transferred to Immobilon®-P PVDF transfer membrane (Millipore, Bedford, MA). After immunoblotting using specific antibodies, proteins were visualized by a PowerOpti-ECL Western blotting Detection reagent (Animal Gentetics, Inc. Korea) and an ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ). Band intensities were quantified using ImageJ software (National Institutes of Health, USA).