JCS018176 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Localization of P-Tyr421-cortactin in mitotic spindle poles. (A) Double-immunofluorescence staining of cortactin with α-tubulin or actin. 3T3-L1 cells at various mitotic phases were stained with anti-cortactin antibody (C) in combination with anti-α-tubulin antibody (T) or anti-actin antibody (A). The nucleus or chromosomes were stained by DAPI (D). M is the merged picture. G1 indicates the interphase cell, and Meta, Ana and Telo indicate the mitotic phases. (B) Immunofluorescence staining of tyrosine phosphorylated cortactin by anti-P-Tyr421-cortactin antibody (PY⁴²¹C) in mitotic 3T3-L1 cell. The fluorescence images were taken with automatic exposure. A background of phospho-cortactin staining can be seen in cell, but the spindle pole staining was over-exposed. (C) The low exposed images of immunofluorescence staining of tyrosine phosphorylated cortactin by anti-phosphotyrosine⁴²¹ cortactin antibody (PY⁴²¹C). The exposure level was set to suit the spindle pole staining by anti-P-Tyr421-cortactin antibody in metaphase. (D) Localization of GFP-cortactin fusion protein in mitotic cell. HeLa cells expressing N-terminal eGFP tagged cortactin were stained with anti-γ-tubulin antibody (γ-Tu) and DAPI (DAPI). The GFP fluorescence was from GFP-cortactin fusion protein. The arrowheads indicate the mitotic spindle poles. Scale bars, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Analysis for specificity of anti-phosphotyrosine⁴²¹ cortactin and anti-P-Tyr466-cortactin antibodies. (A) Construction of GFP-cortactin fusion proteins with mutations on identified tyrosine phosphorylation sites. GFP-Cort wt is the fusion protein of GFP with wild type cortactin; GFP-Cort M1 is the fusion protein of GFP with Tyr466, 475 and 482Phe mutated cortactin; GFP-Cort M2 is the fusion protein of GFP with Tyr421, 475 and 482Phe mutated cortactin; and GFP-Cort M3 is the fusion protein of GFP with Tyr421, 466, 475 and 482Phe mutated cortactin. (B) 3T3-L1 cells were treated with (V) or without (C) 20 µM vanadate for 24 hours, and cortactin protein was immunoprecipitated (IP) by anti-cortactin antibody (α-Cort). The immunoprecipitated samples were blotted (IB) with anti-cortactin antibody (α-Cort) or anti-phosphotyrosine antibody (α-PY). (C) Phosphorylation on Tyr421 and Tyr466 of cortactin in vanadate-treated 3T3-L1 cell. The whole-cell lysates were blotted (IB) using anti-cortactin antibody (α-Cort), anti-P-Tyr421-cortactin antibody (α-PY⁴²¹C) or anti-P-Tyr46621-cortactin antibody (α-PY⁴⁶⁶C). V, vanadate-treated cell; C, control cell. (D) Specificity of anti-P-Tyr421-cortactin and anti-P-Tyr466-cortactin antibodies. Cells were transfected with vector expressing GFP-cortactin fusion protein (panel A) and treated with vanadate to induce tyrosine phosphorylation. The exogenous expressed GFP-cortactins and endogenous cortactin were immunoprecipitated with anti-cortactin antibody and blotted with anti-P-Tyr421-cortactin, anti-P-Tyr466-cortactin, anti-eGFP or anti-cortactin antibodies. Wt, M1, M2, and M3 are as described in panel A. (E) Analysis of anti-P-Tyr421-cortactin and anti-P-Tyr466-cortactin antibodies from Sigma and BioSource. Anti-P-Tyr421-cortactin antibody and anti-P-Tyr466-cortactin antibody from BioSource were characterized and used by Head et al. (Head et al., 2003). Anti-P-Tyr421-cortactin antibody and anti-P-Tyr466-cortactin antibody from Sigma were characterized and used in our present study. Mitotic HeLa cell was stained by anti-P-cortactin antibodies from Sigma as well as BioSource. γ-Tu, anti-γ-tubulin antibody; DAPI, DAPI staining; M, merged picture. Scale bars, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. No centrosomal staining by anti-P-Tyr421-cortactin or anti-P-Tyr466-cortactin antibody in cortactin knockdown 3T3-L1 cells. (A) The small interfering RNA (siRNA) sequence corresponding to cortactin nucleotides at residues 251-271 (S1: 5′-GGGTGCTAAAACCGTGCAGGG-3′) was selected to construct cortactin siRNA plasmid in the pcDNA3/U6 vector. The cortactin knockdown 3T3-L1 cells were stained by anti-cortactin antibody (Cort) and anti-actin antibody (actin). (A) Cortactin protein was reduced in the stable cortactin knockdown cells. Whole-cell lysates from wild-type 3T3-L1 cell (C), control siRNA plasmid transfected stable cell (Sh1, 5′-GGGTAGAGCTAAGCCGTGCAG-3′) and two stable cortactin knockdown cells (Ri1 and Ri2) were blotted by anti-cortactin antibody. (C) The cortactin knockdown 3T3-L1 cells (Ri2) were stained by anti-P-Tyr421-cortactin (PY⁴²¹C) or anti-P-Tyr466-cortactin (PY⁴⁶⁶C) antibodies. The chromosome was stained by DAPI (DAPI) and centrosomes were stained by anti-γ-tubulin antibody (γ-Tu). The arrowheads indicate the centrosomes. (D) The overexposed images in panel C. (E) The dynamic association of activated Src kinase (PY⁴¹⁸Src) with centrosomes/spindle poles during mitosis. 3T3-L1 cells were stained with anti-phosphotyrosine⁴¹⁸ Src antibody (P⁴¹⁸Src) and anti-γ-tubulin antibody (γ-Tu). The mitotic phases were identified by DAPI staining (DAPI). The arrowheads indicate centrosomes/spindle poles. (F) Fluorescence intensity of PY⁴¹⁸-Src and PY⁴²¹-cortactin at centrosomes/spindle poles. The fluorescence intensity was measured by the fluorescence intensity multiplied by the area of antibody staining at centrosomes/spindle poles. The fluorescence intensity was averaged from 10-20 cells and the relative fluorescence intensity was calculated. Scale bars, 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Staining of focal adhesion points by anti-phosphotyrosine⁴²¹ cortactin antibody in wild type 3T3-L1 cell but not in cortactin knockdown 3T3-L1 cell. A: Immunofluorescence staining of tyrosine phosphorylated cortactin at focal adhesion points in 3T3-L1 cell. 3T3-L1 cells were stained by anti-phosphotyrosine⁴²¹ cortactin antibody (PY⁴²¹C), anti-paxillin antibody (PAX) and DAPI (DAPI). M is the merged picture. B: Immunofluorescence staining by anti-phosphotyrosine⁴²¹ cortactin antibody in cortactin knockdown 3T3-L1 cell. The labels are the same as in panel A. Microscopic image for anti-phosphotyrosine⁴²¹ cortactin antibody staining was over-exposed. The cortactin knockdown 3T3-L1 cell was Ri2 as indicated in Fig. S3. C: Constitutively activated Src kinase, but not inactive Src kinase induced co-localization of tyrosine phosphorylated cortactin with paxillin. NIH3T3 cells expressing constitutively activated Src kinase (Phe mutation on Tyr⁵²⁷ in a Src kinase of chicken origin) or inactive Src kinase (Met mutation on Lys²⁹⁵) were double-stained by anti-cortactin antibody (Cort) and anti-paxillin antibody (PAX), anti-phosphotyrosine⁴²¹ cortactin antibody (PY⁴²¹C) and anti-paxillin antibody, or anti-cortactin antibody and anti-phosphotyrosine⁴²¹ cortactin antibody. D: NIH3T3 cells expressing the exogenous eGFP-tagged cortactin fusion protein (GFP-Cort) or control eGFP protein (GFP) were stained by anti-paxillin antibody (PAX) and DAPI (DAPI). eGFP-cortactin was reveal by eGFP fluorescence. The focal adhesion points labeled by paxillin were also labeled by eGFP-tagged cortactin fusion protein but not eGFP. Scale bars, 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Association of tyrosine phosphorylated cortactin with focal adhesion points and mitotic centrosomes in COS 7 cell. A: The mitotic COS 7 cells were stained with anti-γ-tubulin antibody (γ-Tu) for centrosomes/spindle poles and anti-phosphotyrosine⁴²¹ cortactin antibody (PY⁴²¹C) or anti-phosphotyrosine⁴⁶⁶ cortactin antibody (PY⁴⁶⁶C) for phospho-cortactin. G1, G1 phase; G2-M, G2-M transition; Pro, prophase; Meta, metaphase; Ana, anaphase; Telo, telophase. Scale bars, 10 µm. B: Constitutively activated Src kinase induced co-localization of tyrosine phosphorylated cortactin with paxillin in COS 7 cells. COS 7 cells expressing constitutively activated Src kinase (Phe mutation on Tyr⁵²⁷ in a Src kinase of chicken origin) were double-stained by anti-cortactin antibody (Cort) and anti-paxillin antibody (PAX), anti-phosphotyrosine⁴²¹ cortactin antibody (PY⁴²¹C) and anti-paxillin antibody, or anti-cortactin antibody and anti-phosphotyrosine⁴²¹ cortactin antibody. The bar is 50 µm. C: COS 7 cells stained with anti-cortactin antibody (Cort), phalloidin (F-actin) and DAPI (DAPI). M is the merged picture. Scale bars, 50 µm.