Delayed p65 nuclear translocation is T4519 dependent and early p65 nuclear translocation is proteasome dependent in human MoM cells.

MoM cells were infected with wild type S. Typhi or Ty2ΔT4519 for 30 mins followed by gentamycin protection assay as described under Materials and Methods. A. Quantification of IκB-α and β-actin western blots. Quantification was done by using ImageJ software and graph is plotted in graph pad prism 8.B-D–p65 confocal microscopy staining after infection with GFP tagged bacteria. MoM cells were incubated with indicated inhibitors followed by infection. Alexa-fluor conjugated secondary antibody (Alexa-fluor 594 anti rabbit) was used for p65 staining and nucleus was stained by Hoechst (blue). Here NF stands for no Infection. The above experiments were repeated three times and the values from those three experiments were plotted. Error bars represent SD. Significance was calculated using two tailed unpaired T test. Statistical analysis was done by using GraphPad Prism 8. NS stands for not significant. A representative image from each experiment was given. E.Quantification of fluorescence colocalization of Hoechst (blue) and p65 (red), done by taking random fields, from which in a total of 50 cells, blue and red co-localization were measured by using LSM 710 Zeiss Zen Blue software of confocal microscope. P***< 0.001,compared between Ty2 1 hr and MG132 Ty2 1 hr infected cells and P***< 0.001, compared between Ty2ΔT4519 Ty2 1 hr and MG132 Ty2ΔT4519 1 hr infected cells.

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