FIGURE 2.

CD300c signals through FcϵRγ adaptor molecule. A, RBL-2H3 CD300c-2×Myc cells were transiently transfected with control or FcϵRγ siRNAs. Forty-eight hours after transfection a subset of cells were lysed and proteins (2 μg) were run on 15% SDS-PAGE and transferred to PVDF filters. FcϵRγ interference was assessed by Western blot (WB). Values correspond to FcϵRγ expression, which was calculated by quantifying the intensity of FcϵRγ versus actin. Hexosaminidase release was assessed as described. Triplicates were performed for all the stimulations. The result is representative of two independent experiments. B, COS-7 cells were transiently transfected with HA-tagged CD300c WT or mutant forms (E191V and ΔCyto) in combination with FcϵRγ-FLAG. Cell lysates (0.5% CHAPS) were immunoprecipitated with anti-HA.11 mAb and analyzed by 15% SDS-PAGE under reducing conditions. Proteins were transferred to PVDF filter and probed with the indicated antibodies. Whole cell lysates (2%) were included as controls (**, p ≤ 0.01). Error bars represent standard deviation.