Mouse α actin

Equivalent cytosolic and nuclear fractions were resolved in 10% linear mini sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE, 8.0 × 6.5 × 0.1 cm) at 180 V for 60 min. Proteins were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes at 20 V for 40 min with a Semi-dry apparatus (BioRad), and protein transfer was confirmed by Ponceau-S staining. The membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline with Tween-20 (TBST; 50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) and probed with various primary antibodies. Primary antibodies were: in-house produced rabbit anti-reovirus, rabbit α-GAPDH (Cell Signaling, cat#2118), α-ISG15 (Rockland, cat#200-401-438), and α-IFIT (Abcam, cat#ab55837); goat α-Mx1 (Santa Cruz #sc-34128); and mouse α-Actin (Sigma, cat#A5441), and α-STAT1 (Cell Signaling, cat#9176). Appropriate secondary horseradish peroxidase (HRP)-conjugated horse anti-mouse or goat anti-rabbit (Cell Signaling, cat#7076, cat#7074, respectively), or rabbit anti-goat (Zymed, cat#81-1620) were used to detect immune complexes. Bands were developed by enhanced chemiluminescence and imaged with an Alpha Innotech FluorChemQ MultiImage III instrument.

Cells washed with PBS and mouse brains after dissection were solubilized with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄) supplemented with 1 % Triton X-100 (Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich) and then cleared at 14,000 g at 4 °C for 30 min. Protein concentrations were determined using the BCA protein concentration assay as manufacturer’s instructions (Thermo Scientific). Subsequently, proteins were separated by electrophoresis onto 4–20 % SDS-PAGE gels and then transferred onto Immobilon-P membrane. Membranes were incubated 1 h at room temperature (RT) with the following antibodies: rabbit anti-LRRK2 MJFF2 (1:1000, Abcam), rabbit anti-IL1β (1:1000, Santa Cruz), rabbit anti-COX-2 (1:2000, Cayman Chemical), mouse anti-GAPDH (1:2000, Millipore), mouse anti-β-tubulin (1:3000, Sigma-Aldrich), mouse α-actin (1:3000, Sigma-Aldrich), rabbit anti-p65 total (1:2000, Cell signaling), rabbit anti-phospho serine 536 p65 (1:1000, Cell signaling), rabbit anti-p105/p50 (1:2000, Cell signaling), and rabbit anti-phospho serine 337 p50 (1:1000, Santa Cruz). Subsequently, membranes were incubated 1 h at RT with HRP-conjugated secondary antibodies (Sigma-Aldrich) and finally incubated with ECL western blot substrate (Thermo Scientific).