Western bright ecl detection reagents

The total proteins of THP-1 cells infected with Wt, ΔM5447, or Comp strain were prepared at 24 h post-infection, and the concentration of proteins was determined by a BCA Protein Quantification kit (Vazyme). After separation of proteins by SDS-PAGE, the proteins were transferred onto an NC membrane. The membrane was blocked with 5% (w/v) non-fat milk in TBST buffer for 2 h at room temperature and incubated with primary antibodies, NF-κB p65 (Cell Signal Technology, Danvers, MA, United States), pSer536-NF-κB p65 (Cell Signal Technology), or GAPDH (Proteintech), at 4°C overnight. After washing three times with TBST buffer, the membrane was incubated with appropriate HRP-conjugated secondary antibody (Beyotime Biotechnology) for 1 h at room temperature. Finally, the protein bands were visualized by Western Bright ECL detection reagents (Advansta) and quantified using ImageJ software.

The bacterial cells of the Rv1324/Msm strain were inoculated into 200 mL of 7H9 broth and grown with shaking at 37°C until the OD₆₀₀ reached 0.5. The cells were collected and sonicated in lysis buffer at 4°C. Subsequently, the subcellular fractions were harvested via centrifugation.
The preparation of the subcellular fractions from the recombinant Rv1324/Msm was performed as previously described (52 (link)), with modification. Briefly, the cell lysates of Rv1324/Msm were centrifuged at 3,000 × g for 10 min at 4°C to collect the supernatant as whole-cell lysate (WCL). The WCL was then centrifuged at 27,000 × g for 1 h at 4°C to obtain the cell wall pellet (CW), and the soluble (SOL) fraction was then centrifuged at 100,000 × g for 2 h at 4°C to obtain the cell membrane (CM) fraction. The CW and CM fractions were washed once with lysis buffer, recentrifuged, and subsequently resuspended in 0.5 mL of lysis buffer. The concentrations of proteins in the fractions were quantified using a BCA Protein Assay Kit (Vazyme, China), using bovine serum albumin (BSA) as the standard. The fractions were assessed via Western blotting. Finally, the protein bands were visualized using WesternBright ECL detection reagents (Advansta, China).