Journal: Cell Discovery
Article Title: Chronobiology of neurotropic viruses: rhythmic viral entry and arrhythmic host clocks
doi: 10.1038/s41421-026-00867-8
Figure Lengend Snippet: a , b A Venn diagram illustrates the overlap in protein‒protein interactions (PPIs) among Nishigahara G, B19 G, and REV-ERBα ( a ). A PPI network of the Nishigahara G, B19 G, and REV-ERBα proteins. Viral G proteins and REV-ERBα (baits) are shown in yellow. Node sizes correlate with the score from mass spectrometry analyses ( b ). c The binding of RABV G to HUWE1 was confirmed by reciprocal co-IP. d Interaction of G protein from various RABV strains fused with Flag to HUWE1-HA, as determined by Flag-IP and IB with anti-Flag and anti-HA antibodies. e Interaction of RABV G and mutants fused with Flag to HUWE1-HA, as determined by Flag-IP and IB with anti-Flag and anti-HA antibodies. f Immunoblot analysis of extracts from HEK293 cells transfected with REV-ERBα-FLAG and treated with MG132 (10 μM) or overexpressing CVS-11 G for 24 h, followed by treatment with cycloheximide (CHX; 25 μg/mL) for 0 h or 2 h. g Western blot analysis of WT-REV-ERBα-Flag or 55/59SD-REV-ERBα-Flag protein levels in HEK293 cells transfected with EV or CVS-11-G and then treated with 20 mM LiCl for 12 h. h Western blot analysis of WT-REV-ERBα-Flag protein levels in HEK293 cells transfected with EV/WT-CVS-11 G/dCD-CVS-11 G and then treated with 20 mM LiCl for 12 h. i V5-tagged CVS-11 G and HA-tagged REV-ERBα were cotransfected with FLAG-tagged HUWE1 into HEK293 cells, after which the cells were lysed and immunoprecipitated with anti-FLAG antibody and blotted with the indicated antibodies. j , k HA-tagged WT-Ub ( j ) or K48-specific-Ub ( k ) and Flag-tagged REV-ERBα were cotransfected with V5-tagged WT-CVS-11 G or dCD-CVS-11 G and then treated with 20 mM LiCl and 10 μM MG132 for 12 h. The cells were then lysed and immunoprecipitated with anti-FLAG antibody and blotted with the indicated antibodies.
Article Snippet: SR9009 (MedChemExpress, HY-16989), LipofectamineTM 2000 Transfection Reagent (Invitrogen 1,679,991), MG132 (Sigma‒Aldrich, C2211), CHX (MedChemExpress, HY-12320), and LiCl (MedChemExpress, HY-Y0649) were obtained from the indicated suppliers.
Techniques: Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Immunoprecipitation
