Anti actin

Total cellular protein was extracted using RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP 40, 0.5% deoxycholate, 0.1% SDS) supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN). After homogenization with a pellet pestle, the protein cell extracts were centrifuged at 12,000× g for 45 min, and the protein concentration of the supernatant was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins in cell lysates (25 μg) were resolved in 15% SDS–PAGE and electroblotted onto nitrocellulose membranes (Bio-Rad). Nonspecific binding to the membranes was blocked by incubation with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (TTBS) for 1 h at room temperature. Next, membranes were incubated with anti-caspase-3 (1:1000; #9662; Cell Signaling Technology) or anti-ß-actin (1:1000; #3700; Cell Signaling Technology) antibodies in 5% bovine serum albumin in TTBS overnight at 4 °C, followed by the corresponding peroxidase-conjugated anti-rabbit IgG secondary antibody (1:3000; sc-2357; Santa Cruz Biotechnology, Paso Robles, CA, USA) in 5% bovine serum albumin in TTBS for 1 h at room temperature. Peroxidase activity was detected on HyBlot CL autoradiography film (Denville Scientific, Plainfield, NJ, USA). Band intensities were quantified by densitometry using ImageJ software.

Protein samples of 30 μg each were separated on NuPAGE 4–12% Bis-Tris gels (Life Technologies) according to the manufacturer’s instructions. Following separation, proteins were transferred onto PVDF membranes (Immobilion-P, Millipore) using a Mini Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA), at a constant voltage of 100 V for 45 minutes. The membranes were blocked with 5% milk in Tris buffered saline (20mM Tris-HCl, 137mM NaCl pH-7.6) containing 0.1% Tween 20 and then incubated with the respective primary antibody overnight at 4°C. The following primary antibodies were used: anti-BDNF, anti-TrkB and anti-phospho TrkB (Abcam); anti-Akt, anti-phospho Akt, anti-p38 MAPK, anti-phospho p38MAPK and anti-ß-actin (Cell signaling, Boston, MA, USA) all raised in rabbit, 1:1000 dilution. Membranes were washed thoroughly before incubation with the secondary antibody conjugated to horseradish peroxidase enzyme at a 1: 10000 dilution for 1 hour. Immunoreactive bands were visualized by chemiluminescence using SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific). Image Quant was applied to quantify the signals of three independent experiments. The relative band intensities were calculated and normalized to a specific experimental control.

WT, Mavs^−/−, Ifnar^−/−, and Mavs^−/−×Ifnar^−/− DKO BMDCs were infected with WNV at an MOI of 2.5 and harvested and lysed 48 hours later. WT and Nlrp3^−/− BMDC were treated for 30 minutes prior to infection with 25 µg/ml of MAR1-5A3 or GIR-208 [87] (link) and this was maintained throughout the assay. BMDC were lysed in RIPA buffer (10 mM Tris, 150 mM NaCl, 0.02% sodium azide, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS, pH 7.4), with protease inhibitors (Sigma). Samples were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Following transfer of proteins, membranes were blocked with 5% non-fat dried milk and probed with the following panel of primary antibodies: anti-WNV NS3 (R&D Systems), anti-mouse tubulin (Sigma), anti-mouse Ifit2 (gift of Dr. G. Sen, Cleveland, OH), anti-mouse STAT1 (Cell Signaling), anti-mouse IL-1ß (pro and cleaved forms, Abcam), anti-phospho-p65 (Ser536; Cell Signaling), and anti-ß-actin (Cell Signaling).

For western blot analyses, groups of 20–40 embryos at 24 hpf were homogenized by sonication in 50–100 µl of Laemmli buffer. The lysates were separated either on 4–20% Mini-Protean® TGX Stain-Free™ gel or on NuPAGE^TM 4–12% Bis-Tris gels, using Tris-Glycine and MOPS as running buffer, respectively, and transferred to nitrocellulose membranes. The resulting blots were stained using Ponceau, saturated with TBS, 0.1% Tween, and 5% dried milk and probed overnight at 4 °C with Nup133 antibody (Rabbit monoclonal [EPR10809] to NUP133, ab181355; Abcam, 1:500), rabbit polyclonal anti-ß-Actin (Cell Signaling Technology, #4967; 1:1000), or mouse monoclonal antibody HA.11 (clone 16B12; Eurogentec #MMS-101R; 1:2,000). Incubations of the membrane with primary and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were done in TBS buffer (0.1% Tween, 5% dried milk), and signals were detected by enhanced chemiluminescence (SuperSignal® West Femto or Pico PLUS; Thermo Scientific).