(A) Ex vivo expression of DC-STAMP. DC-STAMP−/− cells demonstrate reduced expression of NFATc1 protein in bone marrow, thymus, and spleen. Western blots depict proteins isolated from DC-STAMP +/+ (WT) & DC-STAMP −/− (KO) littermates.
(B) Expression of DC-STAMP in WT and DC-STAMP−/− cells cultured with RANKL and M-CSF. NFATc1 protein expression was induced when DC-STAMP+/+ and DC-STAMP−/− BMMs were cultured in the presence of RANKL+M-CSF. The maximal NFATc1 expression in +/+ and −/− cells was noted at 96 and 48 hours post-infection, respectively. The expression of NFATc1 on DC-STAMP+/+ (left) and DC-STAMP−/− (right) cells after 8, 48, 96 and 120- hours in the presence of RANKL+M-CSF is shown.
(C) Reduced NFATc1 protein level in DC-STAMP−/− cells is restored by DC-STAMP overexpression. Reduced NFATc1 protein expression in DC-STAMP−/− cells (Fig 2B) was restored following retroviral infection with WT DC-STAMP construct. Total proteins were isolated at 4 time points post-infection (48, 72, 96, 120 hours) and subjected to western blot analysis.