Rabbit anti actin

Mouse anti-PRV-UL42 polyclonal antibodies were generated in our lab. Rabbit anti-pSTMN1 was purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-BRAF and Rabbit anti-RPS6KA1 were purchased from Abclonal (Wuhan, China). Rabbit anti-actin was purchased from Proteintech (Wuhan, China). Alexa 555-conjugated goat anti-mouse IgG antibodies were purchased from Millipore (Billerica, MA, USA).

U2OS cells were transfected with the indicated plasmids using XtremeGene360 (Roche). When specified, cells were infected with DENV for the indicated timepoints. Cells were lysed in 1x RIPA + protease inhibitor + 0.1% SDS cocktail (Sigma). Lysates were sonicated prior to being separated by SDS PAGE using a 4-20% Tris-glycine polyacrylamide pre-cast gel (BioRad) and transferred to nitrocellulose membranes. Following 30 min of blocking in PBS + 10% non-fat milk, membranes were probed with the indicated primary antibodies: mouse anti-V5 (Invitrogen), rabbit anti-GFP (Proteintech), mouse anti-GFP (Proteintech), mouse anti-actin (Proteintech, rabbit anti-actin (Proteintech), and rabbit anti-DENV NS3 (GeneTex) followed by near-infrared dye-conjugated secondary antibodies (LiCor) diluted in PBST + 5% non-fat milk and imaged on an Odyssey CLx imaging system (LiCor).