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5 protocols using polyacrylamide tris glycine gels

1

Hippocampal Protein Analysis via Western Blot

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Mice were taken directly from their home cages and killed by cervical dislocation. Whole hippocampi were dissected and frozen in liquid nitrogen. Tissue was homogenized in 200 μL of ice-cold extraction buffer containing PBS, 1 mM EGTA, 1 mM EDTA, 0.01% SDS, and 1 mM PMSF. Protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg for whole cell) were loaded and separated in 10% polyacrylamide Tris–Glycine gels (Invitrogen) and transferred to nitrocellulose membranes using the i-Blot dry transfer system (Invitrogen). Membranes were blocked with Li-Cor blocking buffer. Membranes were incubated for 24 h at 4°C with selective antibodies to: CREB (1:1000; Santa Cruz), pCREB (1:1000, Millipore), TORC1 (1:1000, Cell Signaling), and GAPDH (1:2000, Cell Signaling). Membranes were then incubated with fluorescent secondary antibodies (1:5000, IR-dye 680 or IR-dye 800), before being imaged on an Odyssey fluorescent scanner (Licor Biosciences). GAPDH was used as an internal loading control.
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2

Western Blot Analysis of Synaptic Proteins

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Samples were thawed and homogenized in 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP‐40 (Invitrogen), 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1% Triton X‐100 and phosphatase and protease inhibitor cocktail. Protein concentrations were determined using the BCA kit (Pierce-Thermo Scientific, Rockford, IL). Samples containing 30 µg protein were resolved in 4–20% polyacrylamide Tris–glycine gels (Novex; Invitrogen, Grand Island, NY) and transferred to nitrocellulose membranes at 300 mA for 1 h. Blots were incubated with Odyssey® blocking buffer (Li-Cor; Lincoln, NE) at room temperature for 1 h and were incubated with primary antibody diluted in blocking buffer at 4 °C overnight. Primary antibodies used were anti-PSD-95 (1:1000; Santa Cruz, Dallas, TX), anti-synaptophysin (1:1000; Sigma, St Louis, MO), anti-β-actin (1:15,000; Abcam, Cambridge, UK), anti-cyclophilin (1:10,000; Abcam, Cambridge, UK), anti-amyloid precursor protein (APP) (1:1000 Zymed- Thermo Scientific, Rockford, IL), anti-Bace1 (1:2,000; Milliporesigma, Burlington, MA #5940), and anti-p-AKT-Ser473 (1:1500; Cell Signaling, Danvers, MA). Membranes were incubated with IRDye-conjugated secondary antibodies (1:10,000; LI-COR Biosciences, Lincoln, NE) at room temperature for 1 h, imaged on an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE), and analyzed using NIH Image J.
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3

Western Blot Analysis of IGF1Rα

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Samples were thawed and homogenized in RIPA buffer containing a phosphatase and protease inhibitor cocktail (Thermo Scientific Pierce). Protein concentrations were determined using the BCA kit. Samples containing 30 μg protein were resolved in 15% polyacrylamide Tris-glycine gels (Invitrogen) and electrotransferred to nitrocellulose membranes at 350 mA for 1 h. Blots were blocked with 5% BSA in Tween-TBS at room temperature for 2 h and incubated at 4 °C overnight with polyclonal anti-IGF1Rα antibody (1:500; Santa Cruz) in blocking buffer. Membranes were then incubated with secondary antibody conjugated to IRDye (1:10,000) at room temperature for 2 h, imaged on an Odyssey Imaging System and analyzed using NIH Image J.
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4

Quantification of Immune Signaling Proteins

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The levels of tyrosine-phosphorylated STAT1, RIG-I, IFN regulatory factor 3 (IRF3), and phospho-IRF3 were measured by Western blot analysis as described previously [22 (link),46 (link)]. Whole cell lysates were prepared from virus-infected Calu-3 cultures (MOI = 1) at the indicated time points. Proteins were resolved by electrophoresis on 8% polyacrylamide Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA), and then transferred to polyvinylidene difluoride (PVDF) membranes. The levels of tyrosine-phosphorylated STAT1, RIG-I, IRF3, and phospho-IRF3 were then measured by immunoblotting with mouse monoclonal anti-phospho-Y701-STAT1 Ab, rabbit monoclonal anti-RIG-I Ab, rabbit monoclonal anti-IRF3 Ab, and rabbit monoclonal phospho-IRF3, respectively (Cell Signaling Technology, Beverly, MA, USA). The levels of viral PB1 and M1 proteins in H1N1 viruses carrying silent PB1 and M1 mutations were analyzed by western blot with rabbit anti-PB1 (ThermoFisher Scientific, Rockford, IL, USA) and mouse monoclonal anti-M1 Abs (Abcam, Cambridge, MA, USA), respectively.
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5

Quantifying STAT1 Tyrosine Phosphorylation

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The levels of tyrosine-phosphorylated STAT1 were measured by Western blotting (WB) as described previously (Dickensheets et al, 2013 (link)). NHBE cells were treated with recombinant human IFN-λ1, -λ2, or -λ3 (R&D Systems, Inc.) at 500, 50, or 5 ng/mL for 30 min at 37°C. Whole cell lysates were then prepared and assayed directly by WB or after immunoprecipitation of STAT1 protein using a rabbit anti-STAT1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The protein extracts were resolved by electrophoresis on 8% polyacrylamide Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA), and then transferred to polyvinylidene difluoride membranes. The levels of tyrosine-phosphorylated STAT1 were measured by immunoblotting with mouse monoclonal anti-phospho-Y701-STAT1 Ab (Cell Signaling Technology, Beverly, MA, USA). The levels of total STAT1 protein were measured by reprobing the blots with an anti-STAT1 antibody.
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