Amersham ecl

The concentration of mossMFHR1 used for activity assays was measured via ELISA, using the same antibodies and protocol performed for mossFH as described above. As standard for the ELISA, a batch of purified mossMFHR1 was used in which its concentration was determined via band densitometry (Quantity One, Bio-Rad, Munich, Germany) after SDS–PAGE (Ready Gel Tris-HCl, Bio-Rad) and Coomassie staining. The sandwich ELISA using this MFHR1 standard protein has a linear response between 1 and 64 ng/ml. Electrophoretic separation of proteins was carried out in 10% SDS–polyacrylamide gels (Ready Gel Tris-HCl; BioRad) at 120 V. Subsequently, gels were stained with PageBlue® Protein Staining Solution (PageBlue™, Thermo Fisher Scientific). For Western blot analysis, SDS-PAGE gel was blotted to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA) in a Trans-Blot SD Semi-Dry Electrophoretic Cell (Bio-Rad) for 1 h with 1 mA /cm² membrane. Immunoblotting was performed using mouse anti-His antibodies (MAB050, R&D Systems, Minneapolis, MN, USA) as primary and sheep anti-mouse antibodies coupled to peroxidase (NA931, Amersham ECL™, GE Healthcare) as secondary antibody in a 1:500 and 1:10,000 dilution respectively, followed by chemiluminescence development (ECL™ Advance Western Blotting Detection Kit, GE Healthcare) following the manufacturer’s instructions.

B16-F10 cells (2 × 10⁶ cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).

CEF cells (ca. 1 × 10⁶), infected with rMVA at m.o.i. 5 (or mock-infected) were lysed with sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.1 mg/mL bromophenol blue), and immunoblotting was performed as previously described [9 (link)]. In brief, protein samples were resolved by 10% SDS–PAGE and transferred to nitrocellulose. Reversible Ponceau staining was used as a loading control [27 (link)]. The HAG fusion protein was detected using chicken anti-H1N1 polyclonal antibodies (Istituto Zooprofilattico Sperimentale Tre Venezie, Padova, Italy), and the M1-V5 transgene was detected using the anti-V5 monoclonal antibody V5-10 (Sigma–Aldrich), respectively followed by HRP-conjugated rabbit anti-chicken Ig, or anti-mouse Ig antibodies (DakoCytomation, Denmark A/S). Western blots were developed by enhanced chemiluminescence (Amersham ECL; GE Healthcare, UK) autoradiography.

Cytosolic and nuclear fractions were prepared using the NE‐PER Nuclear and Cytosolic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer's instructions. Sample protein content was determined by the bicinchoninic acid assay protein assay (Thermo Fisher Scientific). Extracts were analysed by SDS–PAGE using a 10% Bis–Tris NuPAGE gels (Invitrogen) and transferred onto PVDF membranes (Millipore). Subsequent to being washed with PBS containing 1% Tween‐20 (PBS‐T), the membranes were blocked in 5% bovine serum albumin (BSA) in TBS‐T for 1 h at RT and then incubated with the primary antibody overnight at 4°C. The membranes were incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature and exposed to X‐ray film (Kodak) after incubation with Thermo Scientific Pierce ECL or Amersham ECL (GE Healthcare). ImageJ software (NIH) was used for the quantification of the bands. All bands were normalised against the loading controls.

Aliquots of protein supernatants containing equal amounts of protein and sodium dodecyl sulphate (SDS) reducing buffer were boiled for 5 min. They were then electrophoresed on SDS-polyacrylamide gels and transferred to polyvinyldiene difluoride membranes. The membranes were blocked with 5 % non-fat dry milk and probed with specific primary antibodies of monoclonal anti-Bax and Bcl2 antibodies followed by incubation with peroxidase-conjugated secondary antibodies. The blots were developed with Amersham ECL western blotting kit (GE Healthcare, Amersham Place, Little Chalfont, U.K) according to the manufacturer’s instructions. The blots were quantified by ChemiDoc XRS 4.6.9 (Bio-Rad Laboratories Inc., Hercules, CA, USA.) software and protein loading was corrected for β-actin as loading control.