Ab45427

Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 150 mM NaCl), supplemented with protease inhibitor cocktail (Roche), 5 mM NaF and 0.2 mM Sodium orthovanadate. Protein lysates were mixed with reducing agent and LDS sample buffer (Novex, ThermoFisher) and denatured at 70°C for 10 min and loaded in Novex Nupage 4–12% Bis-Tris gels. Gels were transferred onto nitrocellulose membrane using the iBlot2 system (ThermoFisher). Membranes were blocked with PBS-0.05% Tween and 5% milk for 1 h at room temperature with agitation, before overnight incubation with primary antibodies. Antibodies against PKR (ab45427 Abcam), ILF3 (ab92355 Abcam), s6RP (2317S CST), eIF6 (3833S CST), ILF2 (ab154169 Abcam), α-tubulin (CP06 Merck), fibrillarin (ab5821 Abcam), phospho-eIF2α (Ser-51) (D9G8) (3398S CST), IFNAR1 (ab10739 Abcam), IFIT3 (ab76818 Abcam), OASL (ab191701), IRF1 (CST #8478), eIF3M (Bethyl, A305–029A), anti-rabbit HRP (CST) and anti-mouse HRP (Bio-Rad) were used. Proteins were visualized using ECL (Pierce) on a Bio-Rad ChemiDoc imaging system. Protein bands were quantified using ImageJ (v1.51p) software and normalized to α-tubulin or fibrillarin.

Brain tissue was collected from newborn and adult mice and homogenized in T-PER tissue protein extraction reagent (78510; ThermoFisher) supplemented with Halt protease and phosphatase inhibitor cocktail (78440; ThermoFisher). Western blot analyses were performed on tissue lysates using antibodies against IFNAR1 (ab124764; Abcam), PKR (ab45427; Abcam), cGAS (ABF124; Millipore), TLR-3 (6961; Cell Signaling), STAT1 (9172; Cell Signaling), and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (ab8245; Abcam) as a loading control. A 1:1,000 dilution was used for all primary antibodies. Blots were visualized using the LI-COR Odyssey system, and densitometric analysis was performed using LI-COR Image Studio Lite 5.2.5.