Figure 1
Figure 1. DEP-1 is tyrosine phosphorylated in a SFK-dependent manner. (A) Structure of full-length DEP-1 and Myc-tagged Myr-DEP-1. (B) Myr-DEP-1 WT and inactive Myr-DEP-1 D/A mutant were expressed in HEK 293 cells. Tyrosine phosphorylation of immunoprecipitated DEP-1 was analyzed by immunoblotting (IB) with the PY99 phosphotyrosine antibody (PY). The membrane was stripped and reprobed with the Myc antibody to detect equivalent levels of immunoprecipitated Myr-DEP-1. (C) Tyrosine phosphorylation of full-length WT DEP-1 is increased by pervanadate (PV) treatment of HEK 293T cells transfected with empty vector (pmT2) or full-length WT DEP-1. At 48 hours after transfection, cells were incubated or not with PV (100μM) for 20 minutes before cell lysis. DEP-1 was immunoprecipitated (IP) with the clone 143-41 mouse antibody, and its tyrosine phosphorylation determined by immunoblotting with the PY99 antibody. The membrane was stripped and reprobed with the DEP-1 antibody (clone 143-41) to show similar levels of immunoprecipitated DEP-1. (D) Tyrosine phosphorylation of Myr-DEP-1 D/A is impaired by PP2 or by a Src dominant-negative mutant. HEK 293 cells were transfected with Myr-DEP-1 D/A and incubated with either PP2 (5μM; 30 minutes) or vehicle (DMSO), or cotransfected with a Src dominant-negative mutant (RF-Src). Tyrosine phosphorylation of immunoprecipitated DEP-1 was detected as described in panel B. Src-RF expression was determined after immunoprecipitation with the mouse Src antibody (GD11 clone) and immunodetection with the rabbit clone 36D10. (E) Tyrosine phosphorylation of DEP-1 is increased by coexpression of a constitutively active Src mutant. HEK 293T cells were transfected with empty vector (pmT2), WT DEP-1, or the C/S mutant in the presence or not of active Src Y527F. Tyrosine phosphorylation of immunoprecipitated DEP-1 was determined as described in panel C. Immunoblotting of total cell lysates with the Src antibody (clone GD11) reveals equivalent Src Y527F expression levels. (F) WT Src and Fyn promote DEP-1 D/A tyrosine phosphorylation. SYF MEFs were transfected with empty vector (pmT2), the DEP-1 D/A mutant alone, or in combination with either WT Src- or Fyn-encoding vectors. Phosphorylation of immunoprecipitated DEP-1 is revealed as described in panel C. Immunoblotting of total cell lysates with the indicated antibodies demonstrates similar amounts of DEP-1 and SFKs in the various conditions. All results are representative of 3 independent experiments.

DEP-1 is tyrosine phosphorylated in a SFK-dependent manner. (A) Structure of full-length DEP-1 and Myc-tagged Myr-DEP-1. (B) Myr-DEP-1 WT and inactive Myr-DEP-1 D/A mutant were expressed in HEK 293 cells. Tyrosine phosphorylation of immunoprecipitated DEP-1 was analyzed by immunoblotting (IB) with the PY99 phosphotyrosine antibody (PY). The membrane was stripped and reprobed with the Myc antibody to detect equivalent levels of immunoprecipitated Myr-DEP-1. (C) Tyrosine phosphorylation of full-length WT DEP-1 is increased by pervanadate (PV) treatment of HEK 293T cells transfected with empty vector (pmT2) or full-length WT DEP-1. At 48 hours after transfection, cells were incubated or not with PV (100μM) for 20 minutes before cell lysis. DEP-1 was immunoprecipitated (IP) with the clone 143-41 mouse antibody, and its tyrosine phosphorylation determined by immunoblotting with the PY99 antibody. The membrane was stripped and reprobed with the DEP-1 antibody (clone 143-41) to show similar levels of immunoprecipitated DEP-1. (D) Tyrosine phosphorylation of Myr-DEP-1 D/A is impaired by PP2 or by a Src dominant-negative mutant. HEK 293 cells were transfected with Myr-DEP-1 D/A and incubated with either PP2 (5μM; 30 minutes) or vehicle (DMSO), or cotransfected with a Src dominant-negative mutant (RF-Src). Tyrosine phosphorylation of immunoprecipitated DEP-1 was detected as described in panel B. Src-RF expression was determined after immunoprecipitation with the mouse Src antibody (GD11 clone) and immunodetection with the rabbit clone 36D10. (E) Tyrosine phosphorylation of DEP-1 is increased by coexpression of a constitutively active Src mutant. HEK 293T cells were transfected with empty vector (pmT2), WT DEP-1, or the C/S mutant in the presence or not of active Src Y527F. Tyrosine phosphorylation of immunoprecipitated DEP-1 was determined as described in panel C. Immunoblotting of total cell lysates with the Src antibody (clone GD11) reveals equivalent Src Y527F expression levels. (F) WT Src and Fyn promote DEP-1 D/A tyrosine phosphorylation. SYF MEFs were transfected with empty vector (pmT2), the DEP-1 D/A mutant alone, or in combination with either WT Src- or Fyn-encoding vectors. Phosphorylation of immunoprecipitated DEP-1 is revealed as described in panel C. Immunoblotting of total cell lysates with the indicated antibodies demonstrates similar amounts of DEP-1 and SFKs in the various conditions. All results are representative of 3 independent experiments.

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