Fig. 1. Src homology 2 domain–containing adaptor protein B (SHB) expression in bone marrow (BM)–derived dendritic cell (BMDC) development. BM cells from C57BL/6 mice were cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/mL) to generate BMDCs. (A) Total RNA was isolated from immature dendritic cells (imDCs) and lipopolysaccharide (LPS)-treated mature dendritic cells (mDCs), and SHB mRNA was assessed from each sample by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) with the Maxime RT-PCR PreMix (iNtRON) and Fast SYBR Green Master Mix (Life Technologies) kits, respectively. (B) The amount of SHB protein expressed in imDCs and mDCs was assessed by Western blot (WB) assay. (C) SHB expression in imDCs and mDCs was assessed by fluorescence-activated cell sorting after intracellular staining. (D, E) Splenic DCs (spDCs) were isolated from mice using a CD11c⁺ isolation kit (Miltenyi Biotech) and treated or not with LPS (200 ng/mL) for 24 hours. Intracellular SHB protein expression in spDCs was assessed by fluorescence-activated cell sorter (D) and WB assay (E). RT-PCR data are shown as the mean±standard deviation of nine samples pooled from three independent experiments.