Immobilon western chemoluminescent hrp

Whole cell extracts were separated on Mini Protean TGX gels and transferred onto nitrocellulose membrane (BioRad®). SINV-specific ascites fluid (ATCC-VR-1248AF) and antibodies specific to actin (Thermo Fisher®, MS-1295-P), GAPDH (Sigma®, G9545), Flag (Sigma®, F1804), NS1 (CTAD, Icahn School of Medicine at Mount Sinai), STAT1a (Santa Cruz, c-111), and RIG-I (Generously provided by Dr. Adolfo Garcia-Sastre, Icahn School of Medicine at Mount Sinai, NY) were used at a dilution of 1:1000 in 5% milk. Russian spring-summer encephalitis virus ascites fluid (ATCC- V558701562) was used for the detection of Langat virus at a dilution of 1:500 in 5% milk. Antibodies specific to GFP (Santa Cruz, sc-8334) and RRV-specific ascites fluid (ATCC-VR-1246AF) were used at a dilution of 1:200 in 5% milk. The SINV nsP4 antibody was a kind gift from Dr. Richard W. Hardy (Indiana University, Bloomington, IN) and was used at a 1:5,000 dilution in 5% milk in 25 mM Tris-HCl [pH 7.4], 137 mM NaCl, 2.7 mM KCl. and 0.1% Tween20. Mouse and rabbit HRP-conjugated secondary antibodies (GE Healthcare®) were used at a 1:2000 dilution in 5% milk. For antibody detection, Immobilon Western Chemoluminescent HRP (Millipore®) and SuperSignal^™ West Femto Maximum Sensitivity (Thermo Fisher®) substrates were used according to the manufacture’s instructions.