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Fig. 4 | Virology Journal

Fig. 4

From: Lithium inhibits NF-κB nuclear translocation and modulate inflammation profiles in Rift valley fever virus-infected Raw 264.7 macrophages

Fig. 4

Effects of lithium on translocation of NF-kB between the cytoplasm and nucleus, and expression levels of IkB. a The cells were seeded at 4 × 105 cells/well in a 6 well plate for 3 h and then inoculated with 1 × 104.8 viral titre/mL for an hour, the excess virus was substituted with fresh media and various lithium concentrations as well as 5 mg/mL LPS for 24 h. After 24 h of inoculation, cells were fixed with 4% paraformaldehyde for an hour, and then permeabilised with (0.1% Triton X-100, 1%BSA) for 30 min. Permeabilised cells were incubated for 60 min with rabbit anti-p65 antibody (1:500). The primary antibody was followed by FITC-labelled goat anti-rabbit secondary Ab incubation for 60 min. Thereafter, nuclear staining with 25µg/mL DAPI was executed for 5min in the dark. Cells were mounted on slides using 50% glycerol and pictures were captured with fluorescent inverted Nikon Ti-E microscope at 20× magnification. b and c In order to determine the translocation quantity of the NF-κB, Raw 264.7 cell were seeded at 1 × 106 cell/mL for 3 h in the T25 cell culture flasks and then inoculated with 1 × 104.8 viral titre/mL for an hour, the excess virus was substituted with fresh media and various lithium concentrations as well as 5 mg/mL LPS for 24 h. This was then followed by isolation of cytoplasmic proteins as well the nucleus proteins and then western blotting assay followed. The pictures were captured with ChemiDoc XRS+ (Bio-RAD, USA). The ChemiDoc XRS+ image lab 5.2.1 software was used to measure band volume (Bio-RAD, USA). c The plot was developed with Graph pad prism-6 software and instat-3 was used to establish the statistical analysis. d In order to determine the expression of Ikb-α protein, Raw 264.7 cell were seeded at 1 × 106 cell/mL for 3 h and then inoculated with 104.8 viral titre/mL for 1 h, and then the excess virus was substituted with a fresh media and lithium concentrations for 12 h. This was then followed by isolation of proteins and then western blotting assay. The ChemiDoc XRS+ image lab 5.2.1 software was used to capture pictures and measure band volume (Bio-RAD, USA)

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