Journal:

Article Title: Protein Kinase C? Activates c-Src and Induces Podosome Formation via AFAP-110

doi: 10.1128/MCB.24.17.7578-7597.2004

Figure Lengend Snippet: Myristoylated PKC alters actin filament integrity in a c-Src-dependent fashion in SYF cells. (Top row) SYF cells were transiently transfected with both GFP-AFAP-110 (a) and pCMV-c-Src and immunolabeled with EC10 antibody (b). (a to d) In the absence of myrPKC, SYF cells have normal morphology and numerous actin stress fibers. (d) Also, c-Src and GFP-AFAP-110 do not appear to colocalize. (Middle row) SYF cells were transiently transfected with both GFP-AFAP-110 (e) and Flag-tagged myrPKC and immunolabeled with anti-Flag antibody (g). (e to h) In the absence of c-Src, SYF cells display well-formed actin filaments and have normal morphology. (Bottom row) SYF cells were transiently transfected with GFP-AFAP-110 (i), c-Src (j) and Flag-tagged myrPKC (k) and immunolabeled with MAb EC10 and anti-Flag antibody. (i to k) In the presence of both c-Src and myrPKC, SYF cells lose actin filament integrity and instead form actin-rich punctate structures (l). Furthermore, GFP-AFAP-110 and c-Src are also observed to colocalize in the presence of myrPKC (l). EC10 was visualized with Cy5 anti-mouse IgG, while anti-Flag polyclonal antibody was visualized with TRITC-anti-rabbit IgG.

Article Snippet: Anti-Src (N-18) and anti-Src (N-16) polyclonal antibodies were purchased from Santa Cruz, while EC10 MAb and recombinant c-Src used in the Src kinase assay were obtained from Upstate Biotechnology.

Techniques: Transfection, Immunolabeling

Journal:

Article Title: Protein Kinase C? Activates c-Src and Induces Podosome Formation via AFAP-110

doi: 10.1128/MCB.24.17.7578-7597.2004

Figure Lengend Snippet: Ability of dominant-positive AFAP-110 to activate c-Src correlates with colocalization of AFAP-110 with c-Src. (A) SYF cells were transiently cotransfected with c-Src and either wild-type GFP-AFAP-110 (a) or dominant-positive GFP-AFAP-110Lzip (e) and immunolabeled with EC10 (b and f) and antiphosphotyrosine (c and g). Cells coexpressing GFP-AFAP-110 and c-Src display immunoreactivity with the phosphospecific antibody equivalent to that of surrounding nontransfected cells (c), as well as numerous GFP-AFAP-110-decorated actin filaments (a). Likewise, no colocalization is observed between GFP-AFAP-110 and c-Src (d). Cells coexpressing GFP-AFAP-110ΔLzip and c-Src display increased immunoreactivity to the phosphospecific antibody (g), as well as complete disruption of actin filament integrity (e). Also, GFP-AFAP-110ΔLzip and c-Src are observed to colocalize (h). Transfection of GFP-AFAP-110ΔLzip alone into SYF cells (i) resulted in no alteration in actin stress fiber integrity (j), as well as no elevation in cellular tyrosine phosphorylation (k), indicating that AFAP-110ΔLzip activates c-Src and not another tyrosine kinase present in SYF cells. (B) SYF/c-Src cells were transiently transfected with GFP-AFAP-110ΔLzip (m) and immunolabeled with TRITC-phalloidin (n) and anticortactin (o). Expression of the Lzip mutant in SYF/c-Src cells results in a loss of actin stress fiber organization and the formation of actin-rich podosome structures (n). Cortactin is also present in these structures ( o and p). (C) SYF cells were transiently cotransfected with c-Src and either GFP-AFAP-11071A/ΔLzip (q) or GFP-AFAP-110180-226/ΔLzip (u) and immunolabeled with EC10 (r and v) and antiphosphotyrosine (s and w). Cells coexpressing c-Src and GFP-AFAP-11071A/ΔLzip display no elevation in cellular tyrosine phosphorylation (s). In the context of the dominant-positive Lzip deletion, GFP-AFAP-11071A/ΔLzip and c-Src are observed to colocalize (t). Cells coexpressing c-Src and GFP-AFAP-110180-226/ΔLzip display no elevation in cellular tyrosine phosphorylation immunoreactivity with the phosphospecific antibody, equivalent to surrounding nontransfected cells (w). In the context of the dominant-positive Lzip deletion, GFP-AFAP-110180-226/ΔLzip and c-Src fail to colocalize (x). EC10 was visualized with Alexa Fluor 546-goat anti-mouse IgG, antiphosphotyrosine was visualized with Alexa Fluor 647-goat anti-rabbit IgG, cortactin was visualized with Alexa Fluor 647-goat anti-mouse IgG, and actin was visualized with TRITC-phalloidin.

Article Snippet: Anti-Src (N-18) and anti-Src (N-16) polyclonal antibodies were purchased from Santa Cruz, while EC10 MAb and recombinant c-Src used in the Src kinase assay were obtained from Upstate Biotechnology.

Techniques: Immunolabeling, Transfection, Expressing, Mutagenesis

Journal:

Article Title: Protein Kinase C? Activates c-Src and Induces Podosome Formation via AFAP-110

doi: 10.1128/MCB.24.17.7578-7597.2004

Figure Lengend Snippet: PMA directs AFAP-110 to colocalize with c-Src, while PKC inhibitors block PMA-induced colocalization. (A) SYF cells were transiently cotransfected with either GFP-AFAP-110 (a), GFP-AFAP-11071A (e), or GFP-AFAP-110Δ180-226 (i) and c-Src, treated with 100 nM PMA for 30 min, and labeled with MAb EC10 (b, f, and j) and antiphosphotyrosine antibody (c, g, and k). Upon PMA stimulation, SYF cells display a loss of actin stress fiber integrity (a), as well as an increase in immunoreactivity with the antiphosphotyrosine antibody (c). PMA treatment also induced colocalization of GFP-AFAP-110 and c-Src (d). Cells cotransfected with GFP-AFAP-11071A (e) and c-Src displayed immunoreactivity with the antiphosphotyrosine antibody equivalent to that of surrounding nontransfected cells (g) in response to PMA treatment. Cells cotransfected with GFP-AFAP-110Δ180-226 (i) and c-Src displayed immunoreactivity with the phosphotyrosine antibody equivalent to that of surrounding nontransfected cells (k) in response to PMA treatment. (B) SYF cells were cotransfected with either GFP-AFAP-110 (m), GFP-AFP-11071A (q), or GFP-AFAP-110Δ180-226 (u) and c-Src and pretreated with 6 μM bisindolylmaleimide [I] for 6 h and 100 nM PMA for 30 min and then fixed and labeled with EC10 MAb (n, r, and v) and antiphosphotyrosine antibody (o, s, and w). In the presence of bisindolylmaleimide [I], PMA treatment of cells cotransfected with GFP-AFAP-110 and c-Src resulted in no changes in actin filament integrity (m). Likewise, bisindolylmaleimide [I] treatment blocked PMA-induced elevation in cellular tyrosine phosphorylation levels (o) and the colocalization of GFP-AFAP-110 and c-Src (p). In the presence of bisindolylmaleimide [I], PMA treatment of cells cotransfected with GFP-AFAP-11071A and c-Src resulted in no elevation in cellular tyrosine phosphoryation (s). Similar results were observed in cells cotransfected with GFP-AFAP-110Δ180-226 and c-Src (u to x). EC10 was visualized with Cy5 anti-mouse IgG, while antiphosphotyrosine was visualized with TRITC-anti-rabbit IgG.

Article Snippet: Anti-Src (N-18) and anti-Src (N-16) polyclonal antibodies were purchased from Santa Cruz, while EC10 MAb and recombinant c-Src used in the Src kinase assay were obtained from Upstate Biotechnology.

Techniques: Blocking Assay, Labeling

Journal:

Article Title: Protein Kinase C? Activates c-Src and Induces Podosome Formation via AFAP-110

doi: 10.1128/MCB.24.17.7578-7597.2004

Figure Lengend Snippet: PMA treatment of CaOV3 cells overexpressing c-Src does not result in a significant increase in cellular tyrosine phosphorylation. (A) CaOV3 cells were transiently transfected with c-Src and then treated with DMSO, 100 nM PMA (for 30 min), or 100 nM PMA (for 30 min) and 6 μM bisindolylmaleimide [I] (for 6 h). After fixation, the cells were labeled with MAb EC10 (a, e, and i), TRITC-phalloidin (b, f, and j), and antiphosphotyrosine antibodies (c, g, and k). DMSO treatment resulted in no change in cellular morphology or actin filament integrity (a and b), and the cells displayed only slight immunoreactivity to the antiphosphotyrosine antibody compared to surrounding nontransfected cells (c). Likewise, treatment with PMA resulted in no change in cellular morphology or actin filament integrity (e and f), as well as immunoreactivity to the antiphosphotyrosine antibody comparable to that with DMSO treatment (g). Pretreatment with bisindolylmaleimide [I] produced similar results (i, j, and k). (B) myr-PKC was transiently transfected into CaOV3 cells and labeled with anti-Flag MAb (m), TRITC-phalloidin (n), and antiphosphotyrosine antibody (o). Expression of myrPKC had no effect on cell morphology or actin filament organization (n). These cells also displayed immunoreactivity to the antiphosphotyrosine antibody equivalent to that of surrounding nontransfected cells (o). EC10 and anti-Flag MAbs were visualized with Alexa Fluor 488-goat anti-mouse IgG, and antiphosphotyrosine was visualized with Alexa Fluor 647-goat anti-rabbit IgG. Actin was visualized by staining it with TRITC-phalloidin.

Article Snippet: Anti-Src (N-18) and anti-Src (N-16) polyclonal antibodies were purchased from Santa Cruz, while EC10 MAb and recombinant c-Src used in the Src kinase assay were obtained from Upstate Biotechnology.

Techniques: Transfection, Labeling, Produced, Expressing, Staining

Journal:

Article Title: Protein Kinase C? Activates c-Src and Induces Podosome Formation via AFAP-110

doi: 10.1128/MCB.24.17.7578-7597.2004

Figure Lengend Snippet: AFAP-110ΔLzip elevates cellular tyrosine phosphorylation and the formation of actin rosette structures in CaOV3 cells. (A) CaOV3 cells were transiently transfected with either GFP-AFAP-110 (a) or GFP-AFAP-110ΔLzip (e) and c-Src (b and f) and immunolabeled with antiphosphotyrosine antibody (c and g). Expression of GFP-AFAP-110 and c-Src resulted in no significant increase in cellular tyrosine phosphorylation (c). Alternatively, expression of GFP-AFAP-110ΔLzip resulted in a significant increase in cellular tyrosine phosphorylation (g), as well as the disruption of actin filament integrity and the formation of actin-rich podosome structures (e [arrow]). (B) CaOV3 cells were transiently transfected with either GFP-AFAP-110 (i) or GFP-AFAP-110ΔLzip (l) and c-Src and immunolabeled with anticortactin antibody (j and m). Expression of GFP-AFAP-110 (i) does not result in the formation of podosomes, as indicated by the diffuse cortactin staining (j). Expression of GFP-AFAP-110ΔLzip results in the alteration of actin stress fiber integrity and the formation of cortactin-rich podosomes (n [arrow]). EC10 was visualized with Alexa Fluor 546-goat anti-mouse IgG, anticortactin antibody was visualized with Alexa Fluor 647-goat anti-mouse IgG, and antiphosphotyrosine was visualized with Alexa Fluor 647-goat anti-rabbit IgG.

Article Snippet: Anti-Src (N-18) and anti-Src (N-16) polyclonal antibodies were purchased from Santa Cruz, while EC10 MAb and recombinant c-Src used in the Src kinase assay were obtained from Upstate Biotechnology.

Techniques: Transfection, Immunolabeling, Expressing, Staining

Journal:

Article Title: Protein Kinase C? Activates c-Src and Induces Podosome Formation via AFAP-110

doi: 10.1128/MCB.24.17.7578-7597.2004

Figure Lengend Snippet: Mutants of AFAP-110 that abolish interactions with c-Src or PKC are able to block the effects of AFAP-110 in response to PMA treatment in CAOV3 cells. (A) CaOV3 cells were transiently cotransfected with GFP-AFAP-11071A (a, e, and i) and c-Src and then treated with DMSO, 100 nM PMA (for 30 min), or 100 nM PMA (for 30 min) and 6 μM bisindolylmaleimide [I] (for 6 h). After fixation, the cells were immunolabeled with EC10 (b, f, and j) and antiphosphotyrosine antibodies (c, g, and k). PMA treatment resulted in normal cell morphology (a, e and i), as well as no change in cellular tyrosine phosphorylation (g) from that observed upon treatment with DMSO (c). Likewise, pretreatment with bisindolylmaleimide [I] and PMA also resulted in no elevation in cellular tyrosine phosphorylation above background (k). Identical results were obtained upon expression of GFP-AFAP-110Δ180-226 and c-Src after treatment with PMA. EC10 was visualized with Alexa Fluor 546-goat anti-mouse IgG, while antiphosphotyrosine antibody was visualized with Alexa Fluor 647-goat anti-rabbit IgG. (B) Same as panel A, except that the dominant-negative mutant, AFAP-110Δ180-226 was transfected.

Article Snippet: Anti-Src (N-18) and anti-Src (N-16) polyclonal antibodies were purchased from Santa Cruz, while EC10 MAb and recombinant c-Src used in the Src kinase assay were obtained from Upstate Biotechnology.

Techniques: Blocking Assay, Immunolabeling, Expressing, Dominant Negative Mutation, Transfection