Figure 3: Analysis of the Pvf/Pvr and ERK mediated cell spreading response in Drosophila S2 cells.

(a) Effects of dsRNA targeting of either Pvr or Pvf 1-3 genes on GBP-dependent ERK activation in S2 cells. S2 cells were used three days after adding dsRNA of each target gene as described in the Methods. Where indicated, 20 nM GBP was added for 3 min. Each value represents the mean±s.d. for four independent determinations. **P<0.01: Significant differences from control (–GBP/–dsRNA) value are indicated by Tukey’s HSD. Full blot was supplied in Supplementary Fig. 11. (b) ERK activation by conditioned medium of Pvf2-over-expressing S2 cells. 0.1, 0.5, 1.0: 10-fold diluted, 2-fold diluted and undiluted conditioned media of S2 cell culture (5 × 10⁶ cells per well) were respectively used. C. medium: medium from cultures of control S2 cells. 1.0+Ab: undiluted conditioned medium preincubated with anti-Pvf2 antibody for 2 h. Each value represents the mean±s.d. for five independent determinations. *P<0.05: Significant difference from control (BSA) value is indicated by Tukey’s HSD. Full blot is supplied in Supplementary Fig. 11. (c) Western blot of Pvf2 protein in incubation medium of S2 cells stimulated by either 20 nM GBP or 100 nM A23187 for 3 min. In the case of EGTA treatment, S2 cells were preincubated with 10 mM EGTA for 10 min before addition of 20 nM GBP. Liquid chromatography mass spectrometry analysis of the immunoreactive band indicated by the arrow (in GBP-treated sample) showed that two determined peptide fragment sequences &#8232;(VPRPEVVHITR and PRPEVVHITR) were found to be completely identical in Pvf2 amino-acid sequence, indicating that this band is Pvf2 precursor protein (see Supplementary Fig. 6).