Supplementary Figure 1: Characterisation of phospho-fgfr-y463 antibody. (A)

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Supplementary Figure 1: Characterisation of phospho-fgfr-y463 antibody. (A) Cells over-expressing hfgfr1-pcdna3 (+) or pcdna3 (-) were stimulated for 10 minutes with 50ng/ml FGF2 and lysates immunoblotted with anti phospho-fgfr-y463. (B) SYF -/- MEFs expressing Src-WT-GFP were treated with 100μM SU5402 for 5 minutes prior to addition of FGF2. (C) MEFs were stimulated with 50ng/ml FGF2 for 30 minutes and immunoreactivity with anti phospho-fgfr-y463 compared with unstimulated cells or in the presence of 100μg/ml of the phosphorylated Y463 peptide. (D) SYF -/- MEFs expressing Src- WT-GFP were stimulated with FGF2. Cells were stained with anti-phospho-fgfr-y463, anti-phospho-fgfr1-y766 (Santa Cruz, CA, USA) or anti-phospho-fgfr1-y653/654 (Invitrogen, Paisley, UK). Scale bars 25μM. Supplementary Figure 2: Active FGFR or Src is not detected prior to FGF2 stimulation. SYF -/- MEFs were serum starved for 24 hours then stained with anti-phospho- Src-Y416 and anti-phospho-fgfr-y463. Broken arrows indicate protein retained in perinuclear region. Scale bars 25μM. Supplementary Figure 3: Scar1 dependent actin disruption influences activation of FGFR. SYF -/- MEFs expressing Src-WT-GFP and myc-scar1 were stimulated with FGF2. Cells were stained with anti-9e10, anti-fgfr1 or anti-phospho-fgfr-y463 antibodies. Scale bars 25μM.

Supplementary Results Characterisation of novel phospho-fgfr-y463 antibody HEK293T cells (which express low levels of endogenous FGFR1) were transfected with either hfgfr1-pcdna3 (+) or pcdna3 (-), and stimulated with FGF2. Immunoblotting with anti-phospho-fgfr-y463 showed that a single immunoreactive species corresponding in mass to human FGFR1 was detected proving that anti-phospho-fgfr-y463 reacts with activated FGFR1 (Supplementary Figure 1A). When SYF -/- MEFs expressing Src-WT-GFP were treated with an FGFR inhibitor SU5402 prior to stimulation with FGF2 activated FGFR could not be detected with anti-phospho-fgfr-y463 (Supplementary Figure 1B). Furthermore quiescent MEFs were stimulated with FGF2 (Supplementary Figure 1C middle and right hand panels) and immunoreactivity with anti phospho-fgfr-y463 compared with unstimulated cells (Supplementary Figure 1C, left hand panel) or in the presence of 100μg/ml of the phosphorylated Y463 peptide. These results show that antiphospho-fgfr-y463 immunoreactivity is dependant upon FGF stimulation and is blocked by phosphopeptide competition. In order to further validate this novel antibody we stained Src- WT-GFP expressing SYF -/- MEFs with other available phospho-specific FGFR1 antibodies. We observed the same pattern of localisation with antibodies against phospho-fgfr1-y766 and phospho-fgfr1-y653/654 as we did with anti-phospho-fgfr-y463 (Supplementary Figure 1D). With both of these antibodies we detected activated FGFR localised to the plasma membrane and to endosomal structures. Upon treatment with dasatinib this activation could no longer be detected and only a low basal level of activated receptor was present at the cell periphery (data not shown).

Supplementary Table 1 Fig. No. Treatment % of 100 cells 1A phospho-fgfr-y463 detected at PM in MEFs FGF2 99 phospho-fgfr-y463 detected at PM in SYF -/- MEFs FGF2 98 1B FGFR1 at PM in SYF -/- MEFs expressing Src-WT FGF2 100 FGFR1 at PM in SYF -/- MEFs expressing Src-WT SFM 32 increase in phospho-fgfr-y463 in SYF -/- MEFs expressing Src-WT FGF2 100 increase in phospho-fgfr-y463 in SYF -/- MEFs expressing Src-WT SFM 28 2A increase in phospho-fgfr-y463 in presence of Src-251-GFP FGF2 0 increase in phospho-fgfr-y463 in presence of Src-Y527F-GFP FGF2 100 2B increase in phospho-fgfr-y463 in presence of Src-WT-GFP FGF2 100 increase in phospho-fgfr-y463 in presence of Src-WT-GFP dasatinib 2 3B increase in phospho-fgfr-y463 in presence of Src-WT-GFP FGF2 100 increase in phospho-fgfr-y463 in presence of Src-WT-GFP in RhoB -/- MEFs FGF2 30 3C increase in phospho-fgfr-y463 in SYF -/- MEFs expressing Src-WT FGF2 100 increase in phospho-fgfr-y463 in SYF -/- MEFs expressing Src-WT FTI + FGF2 24 4A increase in phospho-fgfr-y463 in SYF -/- MEFs expressing Src-WT FGF2 100 increase in phospho-fgfr-y463 in SYF -/- MEFs expressing Src-WT CD + FGF2 17 4B FGFR1 at PM in SYF -/- MEFs expressing Src-WT FGF2 100 FGFR1 at PM in SYF -/- MEFs expressing Src-WT CD + FGF2 98 S3 FGFR1 at PM in SYF -/- MEFs expressing Src-WT in presence of Scar1-S FGF2 100 FGFR1 at PM in SYF -/- MEFs expressing Src-WT in presence of Scar1 FGF2 97 S3 increase in phospho-fgfr-y463 in SYF -/- MEFs in presence of Scar1-S FGF2 100 increase in phospho-fgfr-y463 in SYF -/- MEFs in presence of Scar1 FGF2 25 Supplementary Table 1: Quantitation. 100 cells were counted for each condition and the percentage of cells with each phenotype is shown. Values that are statistically significant (P < 0.001) are in bold.

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