JCS004366 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Figure S1 -

  Fig. S1. Proteomic analysis of cofilin Ser3 phosphorylation. (A) Representative 2D-DIGE gel image displaying the position of the acidic and basic cofilin isoforms identified by MS. (B) Mass spectrum of the tryptic digest from the acidic gel piece obtained by MALDI-TOF MS and matching to Drosophila melanogaster cofilin/twinstar. (C) Enlarged regions of MALDI-TOF MS spectra showing the TiO₂-enriched N-terminal phosphopeptide of Drosophila cofilin, and non-enriched tryptic digests from gel pieces containing the acidic and basic cofilin isoforms. The 80 Da mass difference between the phosphorylated and not-phosphorylated peaks is indicated. (D) Validation of cofilin Ser3 phosphorylation by 2D immunoblotting using a polyclonal antibody against P-cofilin (pSer3 Cofilin). Enlarged and aligned regions of the 2D-DIGE, colloidal Coomassie blue (CCB) and P-cofilin immunoblot images are shown.

• Supplemental Figure S2 -

  Fig. S2. Analysis of TiO₂-enriched cofilin phosphopeptides by LC-MS/MS. TiO₂-enriched phosphopeptides from the acidic and basic cofilin spots separated on 2D gels were analyzed by LC-MS/MS. (A) Single ion chromatograms of the phosphorylated peptide Ac-ApSGVTVSDVCK (m/z 622.8). Upper panel shows the single ion chromatogram obtained from the basic cofilin spot and the lower panel shows the single ion chromatogram obtained from the acidic cofilin spot. The ion is only detected in the lower ion chromatogram. (B) Tandem mass spectrum of the phosphorylated peptide Ac-ApSGVTVSDVCK. The y-ion series is illustrated together with a few b-ion fragments that localise the phosphorylation site to the N-terminal Ser3 residue.

• Supplemental Figure S3 -

  Fig. S3. Insulin-induced phosphorylation and localisation of Akt in S2R⁺ and Kc167 Drosophila cells. (A) Immunoblotting of P-Akt (pAkt), P-S6K (pS6K) and PP-ERK (dpERK) in serum-starved S2R+ cells that were left untreated (Ctrl) or treated for 30 minutes with 10% FCS, conditioned medium (CM), bovine insulin (10 μg/ml), human EGF (200 ng/ml), murine VEGF (50 ng/ml) and human PDGF (125 ng/ml). β-actin and Akt were used to control for differences in loading. (B) P-Akt (pAkt) immunostaining of S2R⁺ cells stimulated with insulin over time. S2R⁺ cells were maintained in serum-free Schneider’s medium for 16 hours prior to treatment with 10 μg/ml bovine insulin for the times indicated. Cells were then fixed and immunostained for pSer505-Akt. Nuclei were visualised by staining with DAPI (C) P-Akt (pAkt) immunostaining of Kc167 cells stimulated with insulin over time. Cells were treated and stained as above.

• Supplemental Figure S4 -

  Fig. S4. PI3K-dependent actin organisation in Kc167 cells and blockade of insulin/PI3K-dependent ruffling in S2R⁺ cells. (A) Kc167 cells in Schneider’s medium supplemented with 10% FCS were treated for 10 minutes with LY294002 (100 μM), wortmannin (100 nM) or vehicle alone (DMSO) before fixing and staining for actin with Rhodamine-phalloidin. (B) PI3K inhibition blocks insulin-dependent membrane ruffling. Images of time-lapse movies of S2R⁺ cells treated with DMSO, LY294002 (100 μM) and wortmannin (100 nM). Cells were filmed in phase-contrast on a time-lapse microscope using a 100× oil-immersion lens. Frames were acquired every 10 seconds for 30 minutes with treatment after 10 minutes of filming (designated 0’ in the figure). Snapshots of timepoints −10’, 0’, 5?, 10’ and 20’ are shown. The corresponding kymographs depict dynamics at the cell edge along the lines indicated in the left-hand images.