JCS001776 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. IRTKS antibody generation and tissue distribution. A polyclonal antibody against IRTKS was generated as described in material and methods. (A) Western blot of C2C12 extract probed with IRTKS antibody. A band of 60 kDa is recognized. Scale to left indicates molecular mass in kDa. (B) 60 kDa band immunoprecipitated from C2C12 extracts excised from gel and analyzed by mass spectrometry, yielding five peptide sequences, three of which matched sequences within mouse IRTKS. Peptide 2 had no matches, while peptide 5 matches exactly to placental growth factor 2 (Accession number AAP36965.1). (C) Western blot of IRTKS immunoprecipitates from mouse tissue extracts probed with IRTKS antibody. Lower band is Ig heavy chain, upper band is IRTKS. (D) IRTKS is a substrate for the insulin receptor. Myc-IRTKS and the insulin receptor β subunit were expressed in COS7 cells (as indicated) and stimulated with insulin where indicated. Cell extracts were immunoprecipitated with anti-myc and precipitates analysed by immunoblotting, probing either with anti-myc or anti-phosphotyrosine. Tyrosine phosphorylation of myc-IRTKS is observed only when co-transfected with the insulin receptor and stimulated with insulin.

• Supplemental Figure S2 -

  Fig. S2. IRSp53+Ct induces similar structures to IRTKS, with co-localise with VASP and vinculin IRSp53+Ct co-localises with F-actin and VASP (A-D, arrow in D) and vinculin (E-H, arrow in H) at sites of cell-cell contact in a similar manner to IRTKS (compare with Fig. 5). Bar, 20 μm.

• Supplemental Figure S3 -

  Fig. S3. IRTKS mutant fails to induce filopodia-like extensions or actin clusters in COS7 cells. Constructs for expression of IRTKS mutant K4E (K141E, K142E, R145E, K146E), IRTKS IMD K4E or wild-type IRTKS IMD were transfected into COS7 cells and visualized as shown, together with phalloidin staining of the F-actin cytoskeleton. (A,B,C) IRTKS full-length K4E mutant; (D,E,F) IRTKS IMD K4E mutant; (G,H,I) The IRTKS IMD with no mutations. Photos are representative of more than 50 cells where no cells with long filopodia-like protrusions or actin clusters were observed for the mutants and over 80% of cells responded similarly to that shown in G, H and I for the wild-type IMD.