Ecl western blot detection kit

The hippocampus and cortex were homogenized in SDS lysis buffer containing a protease inhibitor and phosphatase inhibitor mixture (Sigma-Aldrich). After sonication, lysates were centrifuged at 3,000×g and 4°C for 15 min. Supernatants were collected, and protein concentrations were determined by the Bradford assay (Bio-Rad). The same amounts of proteins were resolved on SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with primary antibodies overnight at 4°C. Rabbit anti-TNFRSF1A (1:1,000, Abcam), anti-CHRNB2 (1:1,000, Abcam), and anti-β-actin (1:10,000, Abcam) were used as the primary antibodies. Immunoblots were visualized by the ECL western blot detection kit (Millipore) and quantified by densitometry and ImageJ software (National Institutes of Health) (Lee et al., 2017 (link)).

Total cell lysates were extracted using a Triton X-100-based lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.5, 5 mM EDTA, 5 mM NaN₃, 10 mM NaF, and 10 mM sodium pyrophosphate) with a protease inhibitor mix and phosphatase inhibitor cocktail I (Sigma) and centrifuged for 10 min at 13,300 rpm. Proteins were resolved using SDS-PAGE and then transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA). After blocking, blots were developed with a series of antibodies against Txnip (MBL International Co, Woburn, MA, USA), Itch, ubiquitin and iNOS (Cell Signaling Technology, Beverly, MA, USA), and thioredoxin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). GAPDH (Millipore Corporation) and β-actin (Santa Cruz Biotechnology) were used as internal controls. Finally, blots were hybridized with HRP-conjugated goat anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology) and developed using an ECL Western blot detection kit (Millipore Corporation) according to the manufacturer's instructions. The band intensity was measured using Image J software (NIH, Bethesda, MD, USA). For IP analysis, cell lysates were incubated with anti-Txnip Ab (5 μg) and protein G-Sepharose beads for 16 h on a roller at 4°C. The beads were isolated and washed by centrifugation followed by Western blot analysis.

Immunoblotting procedures are described elsewhere^{18 (link)}. Briefly, recombinant human IFN-γ or total lysates were separated using SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). After blocking with 5% BSA, cut blots were developed with one thousand dilution of tested serum and the indicated primary Abs. Finally, the blots were hybridized with HRP-conjugated goat anti-human or anti-rabbit IgG and developed using an ECL western blot detection kit (Millipore Corporation). The relative signal intensity of the proteins was quantified using ImageJ software (version 1.41o; W. Rasband, National Institutes of Health, Bethesda, MD, USA).

Protein lysates were prepared by using the solubilizing solution [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM β-glycerolphosphate, and 1 mg/ml leupeptin]. Protein concentration was determined using Bio-Rad protein assay reagent (Hercules, CA, USA). An equal quantity of proteins was separated by 12 or 15% SDS-PAGE and transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membrane was soaked in 10% skim milk (in PBS, pH 7.2, containing 0.1% Tween-20) overnight at 4°C, then incubated with primary antibodies followed by peroxidase-conjugated anti-mouse or anti-rabbit IgG (KPL, Inc., Gaithersburg, MD, USA). The epitope was visualized by an ECL Western blot detection kit (Millipore Corporation, Billerica, MA, USA). Densitometry analysis was performed by using ImageJ software.

The hippocampus and cortex were homogenized in SDS lysis buffer containing a protease inhibitor and phosphatase inhibitor mixture (Sigma-Aldrich). After sonication, lysates were centrifuged at 3,000×g and 4°C for 15 min. Supernatants were collected, and protein concentrations were determined by the Bradford assay (Bio-Rad). The same amounts of proteins were resolved on SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with primary antibodies overnight at 4°C. Rabbit anti-TNFRSF1A (1:1,000, Abcam), anti-CHRNB2 (1:1,000, Abcam), and anti-β-actin (1:10,000, Abcam) were used as the primary antibodies. Immunoblots were visualized by the ECL western blot detection kit (Millipore) and quantified by densitometry and ImageJ software (National Institutes of Health) (Lee et al., 2017 (link)).

HEp-2 cells were infected with HSV-1 at an MOI of 0.01 and were incubated with or without R. tanguticum nanoparticles (350 µg/ml) at intervals of 6, 12, 18, and 24 h post-infection. Cells were harvested in RIPA Lysis Buffer (Biosharp, Hefei, China) and the soluble fraction was then clarified by centrifugation at 12,000 × g for 5 min at 4°C. Equal amounts of protein (40 µg/sample) were then isolated by 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a pre-equilibrated PVDF membrane (Thermo Scientific). Membranes were blocked with 5% BSA for 2 h, rinsed and followed by incubation with anti-ICP4 antibody (Abcam, ab6514, 1:1000), anti-ICP8 antibody (Abcam, ab20194, 1:500) and anti-GAPDH antibody (Tianjin Sungene Biotech KM9002T, Beijing, China, 1:5,000) in 5% BSA at 4°C overnight, respectively. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature and visualized by using an ECL Western Blot Detection Kit (Millipore Corp., Bedford, MA).