Reduced mast cell numbers in Gab2-deficient mice.
(A) Mast cells in the forestomach, glandular stomach, and skin of Gab2+/+ and Gab2−/− mice. Sections were stained with Alcian blue and nuclear Fast red. Granules of mast cells were stained with Alcian blue. We observed at least 10 fields per each sample, and the representative field was shown. Average numbers of mast cells in 1-cm strip sections are shown. (B) Mast cells stained with berberine sulfate in the forestomach, glandular stomach, and skin of Gab2+/+ and Gab2−/− mice. Berberine sulfate recognizes heparin proteoglycan, which is expressed on mast cells in connective tissues. Berberine sulfate-positive cells were observed under a confocal laser microscope (LSM510; Carl Zeiss, Jena, Germany). (C) Expression of c-Kit and FcεRI. c-Kit expression was detected by staining with biotin–anti–c-Kit (2BP) mAb. To evaluate the FcεRI expression, BMMCs (cultured for 4 weeks) were treated with anti–DNP IgE mAb, and the bound IgE was detected with biotinylated anti–mouse IgE mAb and fluorescein isothiocyanate–streptavidin. Gray histograms indicate unstained negative controls. (D) IL-3 or KitL-mediated proliferation of Gab2+/+ and Gab2−/− BMMCs. Mast cells were expanded by culturing bone marrow cells with IL-3 for 4 weeks. After IL-3 starvation, the cells were cultured in the presence of the indicated concentrations of mIL-3 (left panel) or mKitL (right panel) and were pulsed with 0.5 μCi/well of3H-labeled thymidine for the last 16 hours of the 52-hour culture. Cells were collected by an automated cell harvester, and radioactivity of the incorporated 3H-thymidine was determined by a liquid scintillation counter. (E) Biochemical analysis of signal transduction pathways in BMMCs from Gab2+/+ and Gab2−/− mice. BMMCs were stimulated with mKitL (100 ng/mL) for the indicated periods. Cell lysates were immunoprecipitated with anti–Gab2 or anti–Kit antibodies (ACK2) and were subjected to immunoblotting with anti–phosphotyrosine (4G10), anti–Kit, and anti–Gab2 antibodies. Whole lysates were immunoblotted with anti–diphospho ERKs, anti–phospho Akt (S473), anti-ERK2, and anti–Akt antibodies. Results from the immunoblot analysis were quantified by the densitometric scanning of Western blot bands and indicated as relative activity (RA, activity versus the activity in unstimulated Gab2+/+ BMMCs). (F) Reverse transcription–PCR analysis of Gab1 and Gab2 expression. Total RNAs were isolated from Gab2+/+ and Gab2−/− BMMCs and then reverse transcribed. The cDNA was used for PCR with the Gab1- and Gab2-specific primers.
