Skip to main content
. 2023 May 31;64:102769. doi: 10.1016/j.redox.2023.102769

Fig. 5.

Fig. 5

24HC sequesters the viral particles penetrating the MVB/LE and blocks the OSBP-VAPA interaction. A. N2a cells were pretreated with 5 μM 24HC for 12 h, and then infected with VSV (MOI = 10) for 3 h. Cells were analyzed for the co-localization between VSV nucleoprotein (VSV–N) and Rab7. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. B. N2a cells were pretreated with 5 μM 24HC for 12 h, and then infected with RABV (MOI = 10) for 3 h. Cells were analyzed for the co-localization between RABV nucleoprotein (RABV-N) and Rab7. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. C. Co-immunoprecipitation (Co-IP) was performed to detect the effect of 24HC and 25HC treatment on the interaction between OSBP and VAPA. N2a cells treated with ETOH, 24HC (5 μM), or 25HC (5 μM) for 36 h were harvested and lysed, and then pulled down using OSBP antibody as a bait. The precipitated proteins were analyzed by WB. Grey value comparison was calculated using ImageJ software. D. N2a cells were transfected with expression plasmid pCAGGS-FLAG-VAPA (named FLAG-VAPA) for 12 h and then treated with different concentrations of 24HC for 24 h. FLAG antibody was used as bait and the precipitated proteins were analyzed by WB. E. N2a cells were transfected with FLAG-VAPA for 12 h and then treated with 5 μM 24HC for 24 h, cells were fixed and stained for OSBP, FLAG-VAPA, and DAPI. The cells were then observed by confocal microscopy. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. F. N2a cells were transfected with the expression plasmid pCAGGS-HA-OSBP (named HA-OSBP). After 12 h post-transfection, 5 μM 24HC was added to N2a cell culture media for 24 h. Cells were then fixed and stained with HA-OSBP and DAPI, and the Z-stack of confocal microscopy was used to acquire multi-layer images. The multi-layer images were imported into the ZEN software (version 2.3) to obtain a rendered 3D volume image. Scale bar = 10 μm. The Western blot data are representative of at least two independent experiments.