Figure 2: Characterization of GBP-dependent signalling in Drosophila S2 cells.
From: Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP
(a) Q-100 SAX HPLC analysis of the [3H]-labelled inositol phosphates in S2 cells treated with either 50 nM GBP or vehicle for 45 min. [3H]-labelled inositol phosphates were analysed using a 4.6 × 250 mm Q-100 HPLC column (Thomson Instruments, USA) with an in-line detector. (b) Effects of GBP on the level of 1,4,5 IP3 and 1,4,5,6 IP4. Each value represents the mean±s.d. for three independent determinations *P<0.05; **P<0.01: Significant differences from control (0 nM GBP) value are indicated by Tukey’s HSD. (c) Characterization of the nature of GBP-induced intracellular Ca2+ elevation in GCaMP3-expressing S2 cells. Where indicated, either 10 mM EDTA, 10 mM EGTA, 10 mM BAPTA, 10 μM U73122, 10 μM TMB-8, 100 μM APMSF or 10 μM U0126 was added 10 min before 20 nM GBP. Peak Ca2+-dependent fluorescence signals were normalized to control (+GBP/–Additive) values to indicate relative maximum Ca2+ signals. Each value represents the mean±s.d. for three independent determinations. **P<0.01: Significant differences from control (+GBP/–Additive) value are indicated by Tukey’s HSD. (d) The inhibition of GBP-induced intracellular Ca2+ elevation in GCaMP3-expressing S2 cells by dsRNA targeting of Itpr. A 500 bp irrelevant dsRNA from the MEGAscriptTM dsRNA Kit (Life Technologies, USA) served as a negative control. **P<0.01: Significant difference from control (+GBP/–dsRNA) value is indicated by Tukey’s HSD. Other explanations are as in (c). (e) Effects on 3 min of GBP-induced activation of ERK in S2 cells when either calcium chelators (10 mM EDTA, 10 mM EGTA, 10 mM BAPTA) or serine protease inhibitor (100 μM APMSF) was added 10 min before 20 nM GBP. Each value represents the mean±s.d. for five independent determinations. **P<0.01: Significant difference from control (−GBP/–Additive) value is indicated by Tukey’s HSD. Full blot was supplied in Supplementary Fig. 10. (f) Effects of dsRNA targeting of Itpr on GBP-dependent activation of ERK. **P<0.01: Significant differences from control (–GBP/–dsRNA) value are indicated by Tukey’s HSD. Other explanations are as in d. Full blot is supplied in Supplementary Fig. 10.
