Figure 5
Recruitment of 5-LO to the CD40 receptor complex through association with p85 after stimulation in Raji B cells. (A) The lysates from 1 × 107 Raji B cells that were stimulated either with anti-mouse (control) or anti-CD40 Ig for 10 min were tumbled with GammaBind G sepharose beads. The immune complexes for each stimulation condition were then subjected to SDS-PAGE, which was followed by WB with anti-p85, anti-5-LO, and anti-CD40 Ab. The data represent three independent experiments. (B, C) The Raji B cells, either unstimulated (control) or stimulated with anti-CD40 for 15 min, were labeled for endogenous p85 (green) or CD40 (green) along with 5-LO (red) using anti-p85, anti-CD40, and anti-5-LO, followed by FITC- and TRITC-conjugated secondary Ab, respectively. Cell nuclei were labeled using DAPI (blue). Fluorescence was visualized under a confocal microscope. The merged staining patterns of p85-5-LO and CD40-5-LO are shown in the middle panels. The merged staining patterns of all three colors are shown in the far right panels. Representative images from three separate experiments with similar results are shown.
