Proteomic identification of Jak1 as a protein whose expression was decreased during early osteoclast differentiation. (A) BMMs were incubated with 30 ng/mL M-CSF in combination with 200 ng/mL RANKL for 36 hours. At the end of incubation, cell-surface proteins were biotinylated and isolated. The biotinylated proteins were separated on an 8% to 16% SDS gel and visualized by SYPRO Ruby staining. Protein bands showing differential expression in RANKL-stimulated cells were subjected to LC/MS/MS analysis for protein identification. Arrow indicates the protein band in which Jak1 was included. (B) The decreased expression of Jak1 in RANKL-stimulated cells was confirmed by Western blotting. BMMs were cultured with 30 ng/mL M-CSF alone (M) or M-CSF plus 200 ng/mL RANKL (MR) for 36 hours, and total-cell lysates were subjected to SDS-PAGE followed by immunoblotting using anti-Jak1 antibody. Antiactin blot was shown to ensure same loading between the 2 lanes.