Journal:

Article Title: H-Ras Modulates N -Methyl- d -aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity *

doi: 10.1074/jbc.M302389200

Figure Lengend Snippet: a, L(-tk) cells transfected with SrcWT ±H-RasWT were lysed, and 500 μg of total protein was incubated overnight with anti-Src-agarose (5 μg). Immune complexes (lanes 1 and 2) were washed, resolved by SDS-PAGE, and analyzed by Western blotting with anti-Src or anti-H-Ras monoclonal antibodies. Total protein (50 μg) was also included as a control for expression of transfected protein (lanes 3 and 4). n = 3. IP, immunoprecipitates. b, L(-tk) cells transfected with SrcWT and H-RasWT were lysed and normalized with respect to Src by Western blotting. Lysates (~50 μg) from SrcWT and SrcWT/H-RasWT trans-fections were resolved by SDS-PAGE then analyzed by Western blot using anti-Src[pY418], an antibody that recognizes active Src (top panel). A loading control was run simultaneously (bottom panel). n = 3. c, cell lysates (50 μg) were resolved as before and analyzed using anti-phospho NR2A (top panel) and anti-NR2A antibodies (middle panel). The bar histogram represents percentage NR2A phosphorylation normalized to total NR2A ± S.D. n = 3.

Article Snippet: The NR2A-derived peptide RLLEGNFY(PO 3 )GSLFSV corresponding to amino acids 1318 –1331 was generated by SynPep and used to immunize rabbits.

Techniques: Transfection, Incubation, SDS Page, Western Blot, Bioprocessing, Control, Expressing, Phospho-proteomics

Journal:

Article Title: H-Ras Modulates N -Methyl- d -aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity *

doi: 10.1074/jbc.M302389200

Figure Lengend Snippet: a, control (Con, lanes 1 and 2) and Tat-H-Ras (lanes 3 and 4; 1 μm)-treated P2 and S2 fractions (500 μg) were subjected to immunoprecipitation (IP) using anti-NR2A antibodies (5 μg). The resulting immune complexes were resolved by SDS-PAGE and analyzed by Western blotting (WB) using anti-NR2A (top panel) or anti-pY20 (middle panel) antibodies. The bottom panel shows control input (50 μg) before immunoprecipitation, analyzed using anti-actin antibodies. n = 3. b, homogenates were prepared as in a, and protein was analyzed using anti-NR2B (top panel) and anti-actin (bottom panel) antibodies. n = 3.

Article Snippet: The NR2A-derived peptide RLLEGNFY(PO 3 )GSLFSV corresponding to amino acids 1318 –1331 was generated by SynPep and used to immunize rabbits.

Techniques: Control, Immunoprecipitation, SDS Page, Western Blot

Journal:

Article Title: H-Ras Modulates N -Methyl- d -aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity *

doi: 10.1074/jbc.M302389200

Figure Lengend Snippet: Control (lanes 1 and 2) and Tat-H-Ras (lanes 3 and 4; 1 μm)-treated slice homogenates from Fyn −/− (a) and Src −/− (b and c) mice (P2 and S2; 50 μg) were resolved by SDS-PAGE and analyzed by Western blotting using anti-NR2A (a and b; top panels) and anti-NR2B (c; top panel) antibodies. Bar histograms show data normalized to actin (bottom panels) and are plotted as a percentage of control ± S.D. n = 3.

Article Snippet: The NR2A-derived peptide RLLEGNFY(PO 3 )GSLFSV corresponding to amino acids 1318 –1331 was generated by SynPep and used to immunize rabbits.

Techniques: Control, SDS Page, Western Blot

Journal:

Article Title: H-Ras Modulates N -Methyl- d -aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity *

doi: 10.1074/jbc.M302389200

Figure Lengend Snippet: a, Src tyrosine kinase phosphorylates NR2A, allowing its retention in the synaptic membrane. b, inhibition of Src kinase activity by interaction with H-Ras causes a decrease in the phosphorylation of membrane-bound NR2A. Subsequently there is a reduction in NR2A in the synaptic membrane and a concomitant inhibition in NMDA receptor-mediated LTP.

Article Snippet: The NR2A-derived peptide RLLEGNFY(PO 3 )GSLFSV corresponding to amino acids 1318 –1331 was generated by SynPep and used to immunize rabbits.

Techniques: Membrane, Inhibition, Activity Assay, Phospho-proteomics