V 5507

Cryosections (5 μm) from 6 day old wildtype, Sulf1 or Sulf2 deficient newborns were hydrated with PBS for 10 min followed by a blocking step with 0.1% BSA in PBS for 20 min. Subsequently, the slices were incubated with phage-display antibodies (EV3C3, HS4E4 and RB4EA12, 1:1) for 1.5 h at room temperature. After three washing steps each for ten minutes with PBS, slices were incubated with the anti-mouse VSV secondary antibody (1:100; V-5507, Sigma-Aldrich, diluted in 0.1% BSA in PBS) for 1 h at room temperature. After three further washing steps with PBS and incubation with Alexa 488-coupled goat anti-mouse IgG (Invitrogen, 1:200, diluted in 0.1% BSA in PBS) for one hour at room temperature, slices were washed with PBS for three times, rinsed with methanol, dried and mounted with Fluorescent Mounting Medium (Dako Cytomation).

Anti-CPSF5 (1:5000; 10322-1-AP, Proteintech), anti-CPSF6 (1:5000; ab175237, Abcam), anti-CPSF7 (1:2000; 55195-1-AP, Proteintech), anti-RIG-I (1:1000; D14G6, Cell Signaling), anti-MDA5 (1:1000; D74E4, Cell Signaling), anti-IFIT3 (1:2000; 15201-1-AP, Proteintech), anti-ISG15 (1:2000; 15981-1-AP, Proteintech), anti-DDX21 (1:2000; 10528-1-AP, Proteintech), anti-DHX15 (1:2000; 12265-1-AP, Proteintech), anti-CCL2 (1:2000; 66272-1-Ig, Proteintech), anti-IRF3 (1:1,000; sc-33641, Santa Cruz), anti-phosphorylated IRF3 (1:1000; 4D4G, Cell Signaling), anti-TBK1 (1:1000; D1B4, Cell Signaling), anti-phosphorylated TBK1 (1:1000; D52C2, Cell Signaling), anti-VSVG (1:1000; V5507, Sigma), anti-ICP27 (1:1000; ab31631, Abcam), anti-GAPDH (1:20000; 60004-1-Ig, Proteintech), anti-HA (1:5000; clone HA-7, H6533, Sigma), anti-Flag (1:5000; clone M2, F1804, Sigma), Anti-rabbit IgG (H+L), (Alexa Fluor 555 Conjugate) (1:1000, 4413, Cell Signaling), goat anti-mouse IgG-HRP (1:10000; HA1006, HuaBio), goat anti-rabbit IgG-HRP (1:10000; HA1001, HuaBio). The poly(I:C) (LMW) and poly(dA:dT) were purchased from InvivoGen. Lipofectamine 3000 and Lipofectamine RNAiMAX were purchased from Invitrogen. Act D (actinomycin D) was purchased from Sigma.

The primary antibodies α-NS3 GTX12452(1 : 200), α-E s4G2 clone Hb119 (1 : 200), and the secondary antibodies rabbit α-mouse AF555 (A-21427, Invitrogen), goat α-rabbit AF594 (A-11012, Invitrogen) were diluted in a PermWash solution. Primary antibodies α-GAPDH (1 : 4000) (A01622, Genescript), α-Flavivirus Glycoprotein (1 : 1500) (Ab214336, Abcam), α-p24 (1 : 1000) (ab9044, Abcam), α-VSV-Glycoprotein (VSV-G) (1 : 1000) (V-5507, Sigma) and secondary α-goat IgG HRP (1 : 4000) (A8919, Sigma), α-mouse immunoglobulins HRP (P0260, Dako) were diluted on 10 % Non-Fat-Dried Milk in 1X PBS 0.1 % Tween.