Journal: Frontiers in Immunology

Article Title: Splenic SUMO1 controls systemic inflammation in experimental sepsis

doi: 10.3389/fimmu.2023.1200939

Figure Lengend Snippet: Endotoxemia induced a distinct temporal and substrate specificity for splenic SUMO isoforms. Western-blots (top) and densitometric analyses (bottom) of splenic SUMOylation at the indicated post-LPS time points. Bacterial endotoxin induced SUMOylation-1 of (A) p150, (B) p100, (C) p250, and (D) SUMOylation-2/3 of p70 and p260 (smear). Comparative densitometric analyses of (E) SUMO1 and (F) SUMO2/3 at the indicated post-LPS time-points. β-actin Western-blots (bottom panels) were used as an internal control for protein loading. were used as an internal control for protein loading. Western-blots represent experiments repeated at least twice on different days and graphs show mean ± SEM of fold of induction (FI). * P < 0.05 vs Control (n=3/group, two-way ANOVA with Tukey’s post hoc test).

Article Snippet: The membranes were blocked with 5% skimmed milk-TBST for 1 h at 4°C and incubated with antibodies against SUMO1 (1:1,000, PA5-96065, Thermo Scientific, Rockford, IL) or SUMO2/3 (1:1,000, PA5-19418, Thermo Scientific, Rockford, IL) overnight at 4°C.

Techniques: Western Blot

Journal: Journal of Virology

Article Title: NS5 Sumoylation Directs Nuclear Responses That Permit Zika Virus To Persistently Infect Human Brain Microvascular Endothelial Cells

doi: 10.1128/JVI.01086-20

Figure Lengend Snippet: Mutating ZIKV NS5 K252R and DENV3 NS5 K546R enhances colocalization with SUMO-2/3. Lentivirus-transduced hBMECs expressing ZIKV NS5 WT or K252R mutant (A) or DENV3 NS5 WT, K279R, K546R, or K637R mutants (B) were immunostained for NS5 using anti-FLAG antibody and for SUMO-2/3 and visualized by confocal microscopy. Experiments were done in triplicate, repeated at least three times, and representative data are presented. Bars represent 5 μm.

Article Snippet: To examine protein localization from hBMECs constitutively expressing ZIKV and DENV3 NS5 WT and mutants, ∼60% confluent monolayers were lentivirus transduced for 6 h at 37°C and 5% CO 2 , and staining was performed after 48 h using anti-Flag M2 mouse monoclonal antibody (1:100; Millipore Sigma) coincubated with rabbit polyclonal anti-SUMO-1 (1:1,000; 4930; Cell Signaling), anti-SUMO-2/3 (1:1,1000; PA5-19418; Invitrogen), anti-STAT2 (1:300; 44362G; Thermo Fisher), or anti-PML (1:1,000; A301-167A; Bethyl Laboratories) as described above.

Techniques: Expressing, Mutagenesis, Confocal Microscopy