Journal: PLoS ONE

Article Title: Identification of Nucleolin as New ErbB Receptors- Interacting Protein

doi: 10.1371/journal.pone.0002310

Figure Lengend Snippet: (A) COS7 cells were transiently co-transfected with expression vector of each of the ErbB receptors and Myc-Nucleolin. Cell lysates were separated into cytosolic (supernatant) and total membrane (pellet) fractions. The pellet fraction was subjected to immunoprecipitation with anti-Myc (nucleolin) or anti-HA (control) antibodies and immunoblotted with specific ErbBs antibodies. Total pellet and supernatant extracts are shown in the right lanes of each panel. (B) COS7 cells were transiently co-transfected with expression vector of ErbB1 and Myc-Nucleolin. Nuclear and non-nuclear cell extracts were immunoblotted with anti-ErbB1 antibodies. As control for the fractionation purity Blots were reacted with anti-PARP antibodies (as a nuclear marker) and anti-tubulin antibodies (as a cytosolic marker). (C) Cell lysates prepared from SKBR3 cells were subjected to immunoprecipitation with either anti-nucleolin or anti ErbB2 antibodies and immunoblotted with either anti- ErbB2 or anti-nucleolin antibodies as indicated, as a control total cell lysates are shown in the right lane of each panel.

Article Snippet: Polyclonal rabbit anti phosphorylated ErbB1 (Tyr1068) and phosphorylated ErbB2 (Tyr1248) were purchased from Cell Signaling technology.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Fractionation, Marker

Journal: PLoS ONE

Article Title: Identification of Nucleolin as New ErbB Receptors- Interacting Protein

doi: 10.1371/journal.pone.0002310

Figure Lengend Snippet: (A) COS7 cells were transiently co-transfected with expression vector of either ErbB1 or ErbB2 receptors alone or with Myc-Nucleolin. Following 30 min serum deprivation, cells were either untreated or treated with EGF 100 ng/ml for 5 min. Cell lysates were immunoblotted with anti-phosphorylated EGFR or anti-phosphorylated ErbB2 antibodies respectively. As control, lysates were immunoblotted with anti-EGFR or anti-ErbB2 antibodies. Note that at time 0 phosphorylated receptors are detected in cells expressing nucleolin and EGFR or ErbB2. (B) COS7 cells were transiently co-transfected with expression vector of each of the ErbB receptors and Myc-Nucleolin. Following 30 min serum deprivation cells were untreated or treated with either EGF 100 ng/ml or NRG 100 ng/ml for 5 min as indicated. Cell lysates were subjected to immunoprecipitation with anti-phosphotyrosine antibodies (PY20) and immunoblotted with specific ErbB antibodies. Note that in untreated cells, phosphorylated receptor is detected in cells expressing nucleolin and ErbB receptor. The values represent fold induction compared to the receptor levels in untreated cells (1).

Article Snippet: Polyclonal rabbit anti phosphorylated ErbB1 (Tyr1068) and phosphorylated ErbB2 (Tyr1248) were purchased from Cell Signaling technology.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation