Criterion blotter system

Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 4–12% Criterion gels (BioRad, Hercules, California) with MOPS running buffer and transferred to cellulose membranes (GE Healthcare, Little Chalfont, United Kingdom) with the Criterion Blotter system (BioRad). The following antibodies were used: an anti-STrEP-Tag (#34850, Qiagen, Hilden, Germany), anti-LGP2 (NBP1-85348, Novus, Littleton, Colorado), anti-MDA5 (#5321, Cell Signaling, Danvers, Massachusetts and AT113, EnzoLifescience, New York, NY), anti-RIG-I (D14G6, Cell Signaling) or monoclonal anti-β-actin antibody (A5441, Sigma), IRF3 (#11904, Cell Signaling) or phosphor-IRF3 (ab76493, Abcam, Cambridge, UK,). HRP-coupled anti-mouse (NA9310V, GE Healthcare) or anti-rabbit (RPN4301, GE Healthcare) were used as secondary antibodies. Peroxidase activity was visualized with an ECL Plus Western Blotting Detection System (#RPN2132, GE Healthcare). MV intracellular staining was performed with mouse anti-N mAb (clone 25, [Giraudon and Wild, 1981 (link)]) and FITC coupled Goat Anti-mouse Ab (BD Biosciences, Franklin Lakes, New Jersey). For CHIKV, intracellular staining was performed with either FITC-conjugated anti-CHIK.E2 mAB 3E4 (Bréhin et al., 2008 (link)) or anti-dsRNA mAb (J2-1201, Scicons, Szirák, Hungary) followed by anti-mouse-APC antibody (A865, Invitrogen).

To determine the degree to which siRNA knockdown of CHD8 transcript results in decreased CHD8 protein, total protein was isolated using the Illustra triplePrep kit (GE Healthcare; Waukesha, WI, USA) and protein concentration was determined using the DC protein assay (Bio-Rad; Hercules, CA, USA). Total protein (10 μg) was then separated on a 4–20% gradient criterion TGX gel (Bio-Rad) and transferred to a nitrocellulose membrane by capillary transfer at 80 V for 3 h using a Bio-Rad Criterion blotter system. Blots were incubated overnight at 4 °C with anti-CHD8 (catalog no. 7656, Cell Signaling Technology; Danvers, MA, USA) and anti-GAPDH (catalog no. 1228, Cell Signaling Technology) primary antibodies diluted 1:1000 in blocking buffer. Anti-rabbit immunoglobulin G, horseradish-peroxidase-linked secondary antibody (catalog no. 7074, Cell Signaling Technology) was diluted 1:2000 and incubated with the membrane for 1.5 h at room temperature. Signal was developed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific; Waltham, MA, USA) and captured on a BioSpectrum Imaging System (UVP; Upland, CA, USA). The total density ratio of CHD8 to GAPDH was determined using the VisionWorks LS version 6.8 software (UVP) and analyzed by Student's paired t-test for comparison (n=4).

The brain extracts and cell lysates were separated by 4–12% Bis-Tris gels (Thermo Fisher), followed by transfer to nitrocellulose membrane using Criterion blotter system (Bio-Rad). Nitrocellulose membranes were incubated with Intercept blocking buffer (LI-COR) for 1 hr. at room temperature, followed by overnight incubation with rabbit anti-GM2A antibody (1:1000, Proteintech), mouse anti-GAPDH (1:10,000, Proteintech), rabbit anti-HOMER1 (Synaptic Systems, 1:1000), or mouse anti-Vglut2 (Abcam, 1:1000). Membranes were washed 3 times with TBST and incubated with fluorescent dye-conjugated secondary antibodies (anti-mouse or anti-rabbit, 1:10,000, LI-COR) for 1 hr. at room temperature. Membranes were washed 3 times with TBST and then 2 times with TBS, followed by scanning using an Odyssey Infrared Imaging System (LI-COR).

Cells were lysed in urea buffer (8 M Urea, 0.5% Triton-X) and loaded on a 4–20% Criterion TGX precast gel (BioRad). Protein transfer was performed using Criterion Blotter system (BioRad). The following primary antibodies were used for western blotting; ALFY^{21 (link)}; RARα (sc551, Santa Cruz); PML (sc966, Santa Cruz); ULK1 (8054, Cell Signaling); a-tubulin (5168, Sigma). The antibodies were diluted in 5% BSA TBS-0.1% Tween and incubated at 4 °C over-night. HRP-conjugated donkey anti-rabbit and donkey anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) were diluted in 5% Milk TBS-0.1% Tween and incubated for 1 h at room temperature. Image acquisition was performed with a Syngene chemiluminescence imaging system (Syngene).