NF-κB Activation Analysis

For confocal microscopy, Mtb infected MΦs were placed on poly-L-lysine coated coverslips for 15–20 min. The cells were then stimulated with the curdlan (50 μg/ml) for 30 min at 37°C. Later, cells were fixed with PFA (2%) for 10 min, followed by Triton X-100 (0.1%) treatment for 2 min. The samples were then incubated with BSA (2%) for 2 h to block the non-specific sites. Later, cells were incubated with anti-mouse NF-κB p65 Ab (1:400) for 2 h. Subsequently, cells were incubated with Alexa fluor 633-anti-rabbit Ab for 1 h, followed by staining with DAPI dye. Regular washings were performed at each step (Pahari et al., 2016). The cells were imaged through Nikon A1 confocal laser microscope (Nikon, Tokyo, Japan). Data were analyzed using image analysis software, Nikon NIS-AR 4.1 (Nikon, Melville, NY, United States).

For electrophoretic mobility shift assay (EMSA), nuclear extract was prepared from Mtb infected and curdlan stimulated MΦs. After 30 min of stimulation, an equal amount of nuclear extract (3 μg) from each sample was incubated for 20 min/37°C in water bath with [P³²] end labeled duplex oligonucleotides that contains binding site for NF-κB. The DNA–protein complexes were resolved on a native PAGE-gel (7%) by electrophoresis (Pahari et al., 2016). The gel was dried and exposed to screen at RT for 6–12 h and scanned by phosphor-imager scanning screen (Fujifilm, FLA-5000, Tokyo, Japan).