Nuclear extraction kit

Macrophages were infected with MTB (MOI 20:1), and nuclear extracts were prepared using Nuclear Extraction Kit (Bio-Rad Laboratories, Hercules, USA). Protein concentration was determined using bicinchoninic protein assay kit (Thermo Fisher Scientific, Rockford, IL). Proteins were resolved on SDS-polyacrylamide (12%) gels, and were transferred to methanol-activated Immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA) and probed with specific primary antibody. Following incubation with HRP-conjugated secondary antibody, the proteins were detected by chemiluminescence and quantified densitometrically using imageJ software. Bands were normalized to intensity of corresponding bands for Histone-H3.

Briefly, nuclear and cytoplasm extracts were prepared using a nuclear extraction kit (Bio-Rad, Hercules, CA, USA). The samples were loaded onto 10% SDS polyacrylamide gels and electrophoresed (SDS-PAGE). The proteins were transferred onto a nitrocellulose membrane and incubated with rabbit polyclonal antibodies against StAR (sc-25806, 1:800 dilution) (Santa Cruz, CA, USA), YY1 (sc-1703, 1:800 dilution) (Santa Cruz, CA, USA), GAPDH (sc-25778, 1:2000 dilution) (Santa Cruz, CA, USA), and TBP (ab63766, 1:2000 dilution) (Abcam, Cambridge, MA, USA) overnight at 4 °C. Finally, the membrane was incubated in the dark for 1 h with a fluorescent secondary antibody and specific bands were detected via electrochemiluminescence (ECL) assay. The signals of StAR (30 kDa), YY1 (68 kDa), GAPDH (37 kDa), and TBP (38 kDa) were visualized using ECL reagents (Pierce Biotechnology, Inc. Rockford, IL, USA) and then captured using a chemiluminescence and multicolor fluorescence imaging system (Fusion Fx7, Vilber Lourmat, Marne la Vallée Cedex 1, France). Specific bands were analyzed using Gel-Pro Analyzer 4.0 (Media Cybernetics, Silver Spring, MD, USA).