Pvdf membrane

Anti-Gpx2 (sc-54604) and anti-GCLM (sc-22755) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-action (AP0060) and anti-Nrf2 (BS1258) were purchased from Bioworld (Bioworld, USA). Anti-HO-1 (#5853S) were bought from Cell Signaling Technology (USA). Isolation of cell fractions and Western blotting were performed as detailed previously44 (link). Briefly, the extracts were separated by SDS-PAGE and then electrotransferred to PVDF membranes (Perkin Elmer, Northwalk, CT, USA). Membranes were blocked with 1% BSA for 1 h followed by incubation with a primary antibody at 4 °C overnight. Then they were washed and treated with a DyLight 800 labeled secondary antibody at 37 °C for 2 h. The membranes were screened through the odyssey infrared imaging System (LI-COR, Lincoln, Nebraska, USA).

The transfected cells were lysed in 100 μL of a RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, cocktail protease inhibitor (Roche Applied Science)) on ice for 30 min and centrifuged at 15,000 g for 15 min. The supernatant was added with 100 μL of the loading buffer (2% SDS), while the pellet was sufficiently washed with the RIPA buffer for three times and then added with 50 μL of the loading buffer (4% SDS). Equal volume of supernatant and pellet fractions was subjected to SDS-PAGE with 12% acrylamide gel and transferred onto PVDF membranes (PerkinElmer). The mouse monoclonal antibodies against FLAG or Myc, goat anti-actin (Santa Cruz), goat anti-mouse IgG-HRP antibody and rabbit anti-goat IgG-HRP antibody (Jackson Immuno-Research) were used. The proteins were detected by an ECL detection kit (Thermo scientific). The band intensities were quantified by Scion Image software (Scion Corp).

Cells were washed three times with ice-cold PBS and resuspended in 1% Triton X-100 lysis buffer on ice, followed by protein quantification by the Lowry method. Equal amounts of total protein were separated by SDS-PAGE and transferred onto PVDF membranes (PerkinElmer, USA). The membranes were incubated with appropriate primary antibodies at 4°C overnight after blocking for 2 h at room temperature with 5% skimmed milk in trimethyl benzene sulfonyl tetrazole buffer (TBST). After three washes with TBST, the membranes were incubated with secondary antibodies for 30 min at room temperature. Finally, the immunoreactive protein bands were visualized with enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate, USA), followed by imaging on an Electrophoresis Gel Imaging Analysis System (DNR Bio-Imaging Systems, Israel).

B10R macrophages were infected for 0.5 h, 1 h, and 3 h, with similar numbers of viral particles as in footpad injection, or stimulated with LPS (100 ng/ml) for 60 minutes as performed for induction of NF-κB nuclear translocation; uninfected wells served as negative controls. Experiments were performed in triplicate and proteins were extracted as previously described [15 (link)]. Briefly, following cell lysis and protein extraction of infected and uninfected cells, protein contents were dosed by Bradford Assay (Bio-Rad, Mississauga, ON, Canada). Proteins were resuspended in an SDS sample buffer containing bromophenol blue and beta-mercapto-ethanol, separated by SDS-PAGE using 10% acrylamide gels, and transferred to PVDF membranes (Perkin Elmer, Waltham, MA). Membranes were blocked for 1 hour in TBS- 0.05% Tween 20 containing 5% BSA before overnight incubation with the primary antibody (e.g. phospho-p44/42 (ERK 1/2), ERK 1/2, phospho-p38, p38, phospho-IRF3, IRF3, phospho-TBK1/NAK, TBK). Membranes were washed and incubated with the rabbit secondary anti-HRP-conjugated antibody (GE Healthcare, Baie d’Urfe, QC, Canada), and proteins were visualized by ECL Western Blot Detection System (GE Healthcare). USA).

Under deep anesthesia (sodium pentobarbital i.p.; 50 mg/kg), rats were euthanized at 24 h. The striatal region of the brain was rapidly isolated and homogenized, and total proteins were extracted using protein extraction buffer (Mammalian Cell-PE LBTM, Geno Technology, USA) containing protease and phosphatase inhibitors (Complete Mini, Roche Diagnostics, Indianapolis, IN, USA). Lysed tissues were centrifuged at 12,500 rpm for 15 min (at 4 °C), and the supernatant was collected for storage at −80 °C or to perform a Western blot analysis. Proteins were separated by gel electrophoresis, and then electroblotted onto polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Sciences, USA). Membranes were blocked with 5% non-fat milk, and then incubated overnight at 4 °C with the indicated antibodies, including heme oxygenase (HO)-1, Bax, cleaved caspase-3, Beclin-1, LC3-I/II, and p62 (1:10³ dilution) followed by an appropriate secondary antibody (1:2 × 10⁴ dilution) for 1 h at room temperature. Signals were visualized using enhanced chemiluminescent (ECL) detection reagents (PerkinElmer Life Sciences). The membrane was then stripped and reprobed with an antibody specific for β-actin (1:10⁴ dilution) to ensure the accuracy of each loading. Protein expression was quantified by a BioImaging System (Level Biotechnology).