Annexin II expression by HMDMs. (A) Western blot analysis of whole cell lysates of HMDMs maintained in culture and harvested at the time intervals shown. Blots were probed with anti-CD71 IgG, a macrophage-specific marker and monoclonal anti–annexin II IgG. The level of expression relative to day 5 is shown beneath each band. (B) Western blot analysis of annexin II expression by monocytes and HMDMs. Either biotinylated cell surface proteins (lanes 1 and 2) or whole cell lysates (25 μg, lanes3 and 4) from peripheral blood monocytes on day 0 (lanes 1 and 3) or from HMDMs on day 15 (lanes 2 and 4) were examined by immunoblotting with anti-CD71, anti–annexin II, and anti-GAPDH IgG. Human umbilical vein endothelial cell (HUVEC) lysate (lane 5) served as a positive control. Cell surface biotinylated proteins and whole cell lysates were prepared as described in “Materials and methods.” The level of monocyte/macrophage expression of annexin II relative to day 0 is shown for each lane. (C) Northern blot. Total RNA from freshly harvested monocytes (day 0) or from same-donor HMDMs allowed to differentiate in culture for 2 weeks (day 15) was extracted, resolved by agarose gel electrophoresis, transferred to ζ-probe membranes, and incubated with a 32P-random-primed annexin II cDNA probe. The relative intensities of the radiographic annexin II–specific band and the corresponding ethidium bromide–stained 18S rRNA band are shown below each lane.
