Journal:

Article Title: Growth Factor-Independent Proliferation of Erythroid Cells Infected with Friend Spleen Focus-Forming Virus Is Protein Kinase C Dependent but Does Not Require Ras-GTP

doi:

Figure Lengend Snippet: Expression of dominant-negative Ras in uninfected and SFFV-infected HCD-57 cells transfected with H-rasN17. Cell lysates were prepared for Western blot analysis from uninfected and SFFVP-infected HCD-57 cells cotransfected with a vector expressing the dominant-negative H-rasN17 mutant (lanes 2, 4, and 6) or a control vector (lanes 1, 3, and 5) plus the pEGFP-N2 N-terminal fusion vector expressing GFP and the neomycin resistance gene. Cells expressing GFP were purified by FACS and evaluated for H-rasN17 expression 2 days after transfection (lanes 1 to 4) or 3 months after G418 selection (lanes 5 and 6) using an anti-Ras antibody that detects both endogenous and mutant forms of the Ras protein. Protein loading was determined by immunoblotting with an anti-Raf-1 antibody.

Article Snippet: For Raf-1 and MAPK immune complex kinase assays, protein was purified from 1 mg of lysate using anti-Raf-1 or Erk-2 polyclonal (C14) antibodies from Santa Cruz Biotechnology.

Techniques: Expressing, Dominant Negative Mutation, Infection, Transfection, Western Blot, Plasmid Preparation, Mutagenesis, Purification, Selection

Journal:

Article Title: Growth Factor-Independent Proliferation of Erythroid Cells Infected with Friend Spleen Focus-Forming Virus Is Protein Kinase C Dependent but Does Not Require Ras-GTP

doi:

Figure Lengend Snippet: PKC is required for activation of MAPK but not Raf-1 or MEK in uninfected and SFFV-infected HCD-57 cells. Uninfected and SFFV-infected HCD-57 cells were treated with the PKC inhibitor staurosporine (Stauro) (40 nM) or bisindolylmaleimide (Bis) (20 μM) or the PKA inhibitor HA1004 (40 μM). Uninfected HCD-57 cells were then stimulated with Epo for 15 min, while SFFV-infected HCD-57 cells were left unstimulated. Cell lysates were assayed in immune complex assays for MAP kinase activity (A), Raf-1 kinase activity (B), and MEK kinase activity (C). Protein loading was determined by immunoblotting with the appropriate antibody. MEK−, GST-kinase-inactive MEK.

Article Snippet: For Raf-1 and MAPK immune complex kinase assays, protein was purified from 1 mg of lysate using anti-Raf-1 or Erk-2 polyclonal (C14) antibodies from Santa Cruz Biotechnology.

Techniques: Activation Assay, Infection, Activity Assay, Western Blot