Chikungunya Virus Protein Detection

Western blot (WB) analysis was performed essentially as described previously [14 (link)]. Primary antibodies used were rabbit antisera against CHIKV nsP1, CHIKV nsP2 helicase, CHIKV nsP2 protease, CHIKV nsP3 and CHIKV nsP4 [21 (link)], CHIKV capsid protein, SFV capsid protein (all kind gifts from prof. Andres Merits, University of Tartu, Estonia), rabbit polyclonal against eEF2 #2332, mouse monoclonal against STAT-1 (9H2) #9176, mouse monoclonal against Rpb1 CTD (48H) #2629 (all three Cell Signaling Technology), mouse monoclonal against β-actin #A5316; mouse monoclonal FLAG M2 #F1804 (both Sigma), goat polyclonal against cyclophilin B (C15) #sc-20361, mouse monoclonal against ISG15 (F-9) # sc-166755 (both Santa Cruz), rabbit polyclonal against Renilla Luciferase #GTX125851 (GeneTex) and an in-house raised rabbit polyclonal serum a-GFP. Antisera were diluted in 1% casein in phosphate buffered saline containing 0.1% Tween-20 (PBST). Rabbit polyclonal phospho-eEF2 (Thr56) #2331, rabbit monoclonal rps6 #5G10, rabbit polyclonal PKA Cα #4782 and rabbit polyclonal AMPKα #2532 (all Cell Signaling Technology) were diluted in 1% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST). β-actin, cyclophilin B (cypB) or rps6 were used as a loading control. Biotin-conjugated swine-a-rabbit (DAKO) or goat-a-mouse (DAKO) or goat-a-mouse (Invitrogen) or donkey-a-rabbit (Invitrogen), and Cy3-conjugated mouse-a-biotin #200-162-211 (Jackson Immuno Research) were used for fluorescent detection with a Typhoon-9410 scanner (GE Healthcare) or an Alliance Q9 advanced imager (Uvitec).