rabbit polyclonal anti–phospho–erbb-2(y1248  (Cell Signaling Technology Inc)

Journal: The Journal of Clinical Investigation

Article Title: ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

doi: 10.1172/JCI60568

Figure Lengend Snippet: (A) HEK293 cells were transfected with VSV–Plexin-B1 and constitutively active ErbB-2 (ErbB-2 VE) or wild-type ErbB-2 (ErbB-2 WT). After incubation without (–) or with (+) 150 nM Sema4D for 20 minutes, VSV–Plexin-B1 was immunoprecipitated using an anti-VSV antibody, and precipitates were immunoblotted using anti-phosphotyrosine (pTyr) or anti-VSV antibodies. (B–E) HEK293 cells were transfected with VSV–Plexin-B1 as well as Myc-RhoA and FLAG–PDZ-RhoGEF (B), HA-RhoB and FLAG–PDZ-RhoGEF (C), HA-RhoC and FLAG–PDZ-RhoGEF (D), or HA–R-Ras and Rnd1 (E). Where indicated, cells were additionally transfected with constitutively active ErbB-2, wild-type ErbB-2, or a Plexin-B1 deletion construct that lacks the intracellular domain (PlxB1ΔC). After incubation without or with 150 nM Sema4D for 20 minutes, the indicated active Rho isoforms or R-Ras were precipitated (pull-down) as described in Methods, and precipitates were immunoblotted using antibodies directed against the tags of the Rho proteins or R-Ras.

Article Snippet: The following antibodies were used: rabbit polyclonal anti–cleaved caspase-3 (Cell Signaling Technology), rabbit polyclonal anti-CD31 (Abcam), mouse monoclonal anti–ErbB-2 (clone E2-4001, Invitrogen), rabbit polyclonal anti–phospho–ErbB-2(Y1248) (Cell Signaling Technology), rabbit polyclonal anti–phospho-ErbB-2(Y1248) (Sigma-Aldrich), rat monoclonal anti–Mac-3 (clone M3/84, BD Biosciences — Pharmingen), goat polyclonal anti–Plexin-B1 (R&D Systems), mouse monoclonal anti–Plexin-B1 (clone 439512, R&D Systems), rabbit monoclonal anti-RhoA (clone 67B9, Cell Signaling Technology), rabbit polyclonal anti-RhoB (Cell Signaling Technology), rabbit monoclonal anti-RhoC (clone D40E4, Cell Signaling Technology), mouse monoclonal anti–α-tubulin (Sigma-Aldrich), goat polyclonal anti-VSV (Thermo), mouse monoclonal anti-phosphotyrosine (clone 4G10, Upstate, Millipore), mouse monoclonal anti-FLAG (clone M2, Sigma-Aldrich), rabbit polyclonal anti-Myc (Sigma-Aldrich), mouse monoclonal anti-HA (clone HA-7, Sigma-Aldrich), and trastuzumab (Genentech).

Techniques: Transfection, Incubation, Immunoprecipitation, Construct

Journal: The Journal of Clinical Investigation

Article Title: ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

doi: 10.1172/JCI60568

Figure Lengend Snippet: (A) Human breast cancer cell lines MCF-7, T-47D, SK-BR-3, or BT-474 or (B) BT-474 cells transfected with control siRNA or siRNA against ErbB-2 were lysed; Plexin-B1 was immunoprecipitated; and precipitates were immunoblotted using anti-phosphotyrosine or anti–Plexin-B1 antibodies. In a parallel experiment, levels of active RhoA/RhoC were determined. (C–F) BT-474 cells were transfected with control or Plexin-B1 siRNA. (C) The amount of Plexin-B1 and active RhoA/RhoC was determined. (D) Cell lysates were probed with an anti–phospho–ErbB-2(Y1248) antibody. (E) BT-474 cells were counted on 5 consecutive days. (F) Cells were seeded onto Matrigel-coated filters, and invading cells were counted as described in Methods. (G and H) BT-474 cells stably expressing siRNA-insensitive wild-type Plexin-B1 or siRNA-insensitive mutant Plexin-B1(Y1708F/Y1732F) were transfected with Plexin-B1 siRNA to knock down endogenous Plexin-B1. (G) Plexin-B1 was immunoprecipitated, and precipitates were immunoblotted using anti–Plexin-B1 and anti–phosphotyrosine antibodies. In addition, levels of active RhoA/RhoC were determined. (H) In parallel, cells were seeded onto Matrigel-coated filters, and invading cells were counted. (I and J) BT-474 cells were incubated (I) without or with a mouse monoclonal anti–Plexin-B1 antibody (anti-PlxB1; clone #93, 1.8 ng/μl) or (J) without or with 150 nM PlxB1ext, and the amounts of active RhoA/RhoC were determined. (K and L) BT-474 cells were seeded onto Matrigel-coated filters in (K) the absence or presence of a mouse monoclonal anti–Plexin-B1 antibody (anti-PlxB1; clone #93, 1.8 ng/μl) or (L) the presence of 150 nM PlxB1ext, 2 μg/ml trastuzumab, or both, and invading cells were counted. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used: rabbit polyclonal anti–cleaved caspase-3 (Cell Signaling Technology), rabbit polyclonal anti-CD31 (Abcam), mouse monoclonal anti–ErbB-2 (clone E2-4001, Invitrogen), rabbit polyclonal anti–phospho–ErbB-2(Y1248) (Cell Signaling Technology), rabbit polyclonal anti–phospho-ErbB-2(Y1248) (Sigma-Aldrich), rat monoclonal anti–Mac-3 (clone M3/84, BD Biosciences — Pharmingen), goat polyclonal anti–Plexin-B1 (R&D Systems), mouse monoclonal anti–Plexin-B1 (clone 439512, R&D Systems), rabbit monoclonal anti-RhoA (clone 67B9, Cell Signaling Technology), rabbit polyclonal anti-RhoB (Cell Signaling Technology), rabbit monoclonal anti-RhoC (clone D40E4, Cell Signaling Technology), mouse monoclonal anti–α-tubulin (Sigma-Aldrich), goat polyclonal anti-VSV (Thermo), mouse monoclonal anti-phosphotyrosine (clone 4G10, Upstate, Millipore), mouse monoclonal anti-FLAG (clone M2, Sigma-Aldrich), rabbit polyclonal anti-Myc (Sigma-Aldrich), mouse monoclonal anti-HA (clone HA-7, Sigma-Aldrich), and trastuzumab (Genentech).

Techniques: Transfection, Immunoprecipitation, Stable Transfection, Expressing, Mutagenesis, Incubation

Journal: The Journal of Clinical Investigation

Article Title: ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

doi: 10.1172/JCI60568

Figure Lengend Snippet: (A) RNA of tumor specimens from breast cancer patients without detectable ErbB-2 expression (ErbB-2 score 0) or with ErbB-2 overexpression (ErbB-2 score 3+) was isolated and reverse transcribed. PCR analysis was performed using primers specific for Plexin-B1. (B) Immunohistochemical staining of human breast cancer tissues shows that Plexin-B1 protein is expressed in cancer cells. The staining can be blocked by preincubation of the anti–Plexin-B1 antibody (R&D Systems) with the peptide used for immunization. Scale bars: 50 μm. (C) Breast cancer tissues from 18 different patients without detectable ErbB-2 expression (ErbB-2 score 0) or different levels of ErbB-2 expression (ErbB-2 score 1+ to 3+) were lysed. Plexin-B1 was immunoprecipitated, and precipitates were immunoblotted using anti-phosphotyrosine or anti–Plexin-B1 antibodies. Lysates were probed for ErbB-2, phospho–ErbB-2(Y1248), and α-tubulin. (D) Kaplan-Meier graph representing the disease-free survival of patients with ErbB-2–overexpressing breast cancer. High Plexin-B1 expression, n = 39; low Plexin-B1 expression, n = 22. (E) Kaplan-Meier graph representing the overall survival of patients with ErbB-2–overexpressing breast cancer. High Plexin-B1 expression, n = 13; low Plexin-B1 expression, n = 7. (F) Schematic illustration of the ErbB-2/Plexin-B1 signaling pathway. Overexpression of the receptor tyrosine kinase ErbB-2 results in phosphorylation of Plexin-B1 at two specific tyrosine residues. This phosphorylation of Plexin-B1 promotes the activation of RhoA and RhoC via RhoGEF 11 (PDZ-RhoGEF) and RhoGEF 12 (LARG), which stably interact with the C terminus of Plexin-B1.

Article Snippet: The following antibodies were used: rabbit polyclonal anti–cleaved caspase-3 (Cell Signaling Technology), rabbit polyclonal anti-CD31 (Abcam), mouse monoclonal anti–ErbB-2 (clone E2-4001, Invitrogen), rabbit polyclonal anti–phospho–ErbB-2(Y1248) (Cell Signaling Technology), rabbit polyclonal anti–phospho-ErbB-2(Y1248) (Sigma-Aldrich), rat monoclonal anti–Mac-3 (clone M3/84, BD Biosciences — Pharmingen), goat polyclonal anti–Plexin-B1 (R&D Systems), mouse monoclonal anti–Plexin-B1 (clone 439512, R&D Systems), rabbit monoclonal anti-RhoA (clone 67B9, Cell Signaling Technology), rabbit polyclonal anti-RhoB (Cell Signaling Technology), rabbit monoclonal anti-RhoC (clone D40E4, Cell Signaling Technology), mouse monoclonal anti–α-tubulin (Sigma-Aldrich), goat polyclonal anti-VSV (Thermo), mouse monoclonal anti-phosphotyrosine (clone 4G10, Upstate, Millipore), mouse monoclonal anti-FLAG (clone M2, Sigma-Aldrich), rabbit polyclonal anti-Myc (Sigma-Aldrich), mouse monoclonal anti-HA (clone HA-7, Sigma-Aldrich), and trastuzumab (Genentech).

Techniques: Expressing, Over Expression, Isolation, Immunohistochemical staining, Staining, Immunoprecipitation, Activation Assay, Stable Transfection