Fig. 3:

Generation of IP ₃ in primary B cells: A) Timecourse of IP₃ production in primary B cells. 3x10⁶ MACS-enriched B cells were labeled with ³[H]inositol (60 mCi/ml) for 4 h, then incubated with PBS (non-stimulated) or stimulated with 20 μg/ml anti-IgM for the indicated times. Production of soluble IP species was measured by HPLC and the position corresponding to each species is indicated. IP₃ production was measured as the total counts detected within the 14.25-15.25 minute time window. HPLC profile of an ipk1-5 mutant yeast strain (26) serves as reference. (B): IP₃ production from the B cells in (A) is expressed as the counts in the position corresponding to IP₃. (C): 1x10⁶ FACS-purified wt T1 and T2 B cells were labeled with ³[H]inositol as in (A) and stimulated with 20 μg/ml anti-IgM for 1 minute. IP₃ production was detected as in (A). Data is a representative of four independent experiments. (D): IP₃ production from the T1 and T2 B cells in (C) is expressed as the counts in the position corresponding to IP₃. (E) Fold change in IP₃ level from T1 and T2 B cells following stimulation with anti-IgM was calculated from the counts measured in the position corresponding to IP₃ as in (C and D) (n=3).