Receptor expression levels were detected in total protein extracts from THP-1 cells and control HEK-Blue-null1 and HEK-Blue-null2 cells by anti-NOD1 (Thermo Scientific, USA), anti-NOD2 (Santa Cruz Biotechnology, USA), anti-Dectin-1, and anti-Mincle (InvivoGen, USA) primary antibodies. Anti-GAPDH antibodies (Santa Cruz Biotechnology, USA) were used for sample normalization.
For verification of NF-κB activation after PRR stimulation, THP-1 cells were treated for 30 min with TDB (10 µg/mL) and L18-MDP (100 ng/mL) alone or in combination. Nuclear and cytoplasmic cellular extracts were prepared using an NE-PER kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and normalized using anti-lamin and anti-β-actin antibodies, respectively. The p65 subunit of NF-κB was detected using anti-p65 antibodies (all, Santa Cruz Biotechnology, USA). HRP-conjugated secondary anti-rabbit and anti-mouse antibodies were obtained from GE Healthcare (USA).
For verification of NF-κB activation after PRR stimulation, THP-1 cells were treated for 30 min with TDB (10 µg/mL) and L18-MDP (100 ng/mL) alone or in combination. Nuclear and cytoplasmic cellular extracts were prepared using an NE-PER kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and normalized using anti-lamin and anti-β-actin antibodies, respectively. The p65 subunit of NF-κB was detected using anti-p65 antibodies (all, Santa Cruz Biotechnology, USA). HRP-conjugated secondary anti-rabbit and anti-mouse antibodies were obtained from GE Healthcare (USA).