Westernbreeze chromogenic kit

Cell lysates were prepared in standard NP-40 lysis buffer (Abcam, USA) supplemented with proteinase and phosphatase inhibitors. Protein lysates were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal masses of total protein were separated on 4–12% SDS-polyacrylamide mini-gels and blotted onto PVDF membranes (Millipore, Burlington, MA, USA). Membranes were subsequently blocked, incubated with primary antibodies, and incubated with secondary antibodies, according to WesternBreeze Chromogenic Kit (Thermo Scientific, Waltham, MA, USA). Alkaline phosphatase was detected on the PVDF membranes using a ready-to-use BCIP/NBT substrate (Thermo Scientific, USA) for ready visualization of enzyme-linked antibodies. Rabbit anti-PI3K, anti-phospho-PI3K, anti-ERK, anti-phospho-ERK, and anti-GAPDH antibodies were obtained from Cell Signaling (USA). Quantification of relative intensities was achieved by ImageJ analysis.

The protein level of heat shock proteins (HSP60 and HSP70) and minichromosome maintenance complex 2 (MCM2) was measured after 24 and 72 h of HeLa cells incubation with 50 µg/mL derivatives. Samples corresponding to 100 µg of protein were separated on a 10% SDS/PAGE under reducing conditions and transferred to 0.4 µm (Millipore) polyvinylidene difluoride (PVDF) membrane in a wet transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked with the blocking solution included in the Western Breeze Chromogenic kit (Invitrogen) for 30 min at room temperature. Detection was performed with mouse monoclonal anti-HSP60, anti-HSP70, and anti-MCM2 primary antibodies (1:250 dilution Santa Cruz Biotechnology). Further, the membranes were processed according to the manufacturer’s instructions, using alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies and 5-bromo-4-chloro-3′-indole phosphate/nitroblue tetrazolium as a chromogenic substrate (Invitrogen by Thermo Fisher Scientific). The resulting bands were visualized and photographed using a transilluminator (ChemiDoc MP Video Documentation System, Bio-Rad) and were densitometered using the GelQuant.NET software version 1.7.8.

The generation of polyclonal rabbit antiserum against prokaryotic-expressed rgpTNF-α has been described earlier [18 (link)]. In brief, the rgpTNF-α was mixed with the adjuvant TiterMax Gold (CytRx Corp, Norcross, GA) and injected subcutaneously into New Zealand white rabbits in four injections that were spaced at 3-week intervals. The animals were exsanguinated 5 weeks following the last booster and sera were collected by centrifugation of blood at 1500 rcf for 20 minutes. The serum was aliquoted and stored at −80°C until it was used in the Western blot assay.
Approximately 200 ng of eukaryotic-expressed rgpTNF-α protein was run on a 10–20% tricine gel (Invitrogen) and blotted onto a nitrocellulose membrane using the semidry Minitrans blot electrophoretic transfer cell apparatus (Bio-Rad). The identity of the eukaryotic-expressed rgpTNF-α was determined by its reaction to the polyclonal anti-rgpTNF-α antiserum by Western blot analysis using the WesternBreeze chromogenic kit (Invitrogen) following the manufacturer's instructions with anti-rabbit (IgG) antibody as the secondary antibody (Invitrogen).

The protein level of p53, Beclin-1, LC-3, and Nrf-2 was determined in the samples collected from the control and 100 mg of SiQD/kg of b.w. after 1, 6, 24, and 72 h. Cell lysates corresponding to 100 µg of protein were separated on a 10% SDS/PAGE under reducing conditions and transferred to 0.4 µm polyvinylidene difluoride (PVDF) membrane in a wet transfer tank (Bio-Rad, Hercules, CA, USA). Membranes were blocked with the blocking solution included in the WesternBreeze Chromogenic kit (Invitrogen, Rockford, IL, USA) for 30 min at room temperature. The detection of p53, LC-3, and Nrf-2 proteins was performed with the rabbit polyclonal anti-p53, anti-LC-3, and anti-Nrf-2 primary antibodies, respectively (1:250 dilution). The highlight of Beclin-1 protein was performed using anti-Beclin-1 mouse polyclonal primary antibody (1:250 dilution, SantaCruz Biotechnology, Dallas, TX, USA). After the incubation with primary antibodies, membranes were processed according to the manufacturer’s instructions, using alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies and 5-bromo-4-chloro-3′-indolephosphate/nitroblue tetrazolium as the chromogenic substrate. The resulting bands were visualized and photographed using a transilluminator (ChemiDoc MP Video Documentation System, Bio-Rad, Hercules, CA, USA) and were analyzed using the GelQuant.NET software version 1.7.8.

Tissue or EC samples were homogenised in lysis buffer. Approximately 40μg of tissue protein from VP or 10μg EC protein from lung was separated by 12% SDS-PAGE, transferred to a PVDF Membrane (WesternBreeze Chromogenic Western Blot, Invitrogen, Life Technologies, CA, USA), and incubated with 1:500 rabbit anti-AQP1 (Biozol, Hamburg, Germany) overnight (4°C). Samples were treated with alkaline phosphatase-conjugated anti-rabbit secondary antibody, and immunoreactivity was detected by using the WesternBreeze® Chromogenic Kit according to the manufacturer’s specifications (WesternBreeze Chromogenic Western Blot, Invitrogen, Life Technologies, CA, USA). The intensity of the resulting bands was quantified by densitometry using Image J.

The Protean 3D program (DNASTAR, Madison, WI, USA) was used to predict that an 81-amino acid segment of HPgV-2 NS4A/4B (derived from GenBank accession number KT427414.1) would be localized to the cytoplasm. This region was chosen for expression in the pMAL-C5X vector, which, under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter, allowed for the addition of a maltose-binding protein (MBP) tag at the amino terminus and a 6-histidine tag at the carboxyl terminus (Genscript, Piscataway, NJ, USA). The construct was expressed in Escherichia coli strain BL21(DE3). Following IPTG induction for 4 h at 37°C, cells were lysed, and soluble protein was purified using the ProBond purification system (Invitrogen, Grand Island, NY, USA). Western blotting of the purified protein was performed using a WesternBreeze chromogenic kit (Invitrogen), and purified protein was detected using an anti-His antibody (Invitrogen). Protein was visualized using 5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium (NBT) staining (Novex by Life Technologies) and a Bio-Rad Gel Doc EZ imager, using Image Lab v4.0 software.