modfit mathematical model v4.1.7  (Verity Software House)

Journal: PLoS ONE

Article Title: Defining the Interactions and Role of DCAF1/VPRBP in the DDB1-Cullin4A E3 Ubiquitin Ligase Complex Engaged by HIV-1 Vpr to Induce a G 2 Cell Cycle Arrest

doi: 10.1371/journal.pone.0089195

Figure Lengend Snippet: A-B. HEK293T cells were mock-transfected (lanes 2 and 3) or transfected with HA-Vpr-expressing plasmid (lanes 4 and 5) or transfected with DCAF1 bp3R (DCAF1 1-1507 bp3 siRNA resistant) (lanes 6 and 7), Myc-DCAF1 WD (lanes 8 and 9) or Myc-DCAF1 1377 (lanes 10-11)-encoding plasmids in the presence of HA-Vpr-expressing plasmid. All cells were also transfected with a plasmid encoding GFP and treated with either non-targeting siRNA ( NT siRNA ) (lanes 2, 4, 6, 8 and 10) or specific DCAF1 bp3 siRNA ( bp3 siRNA ) (lanes 3, 5, 7, 9 and 11). Non-transfected HEK293T cells were used as negative control (lane 1). A. Non-transfected or transfected HEK293T cells were lysed in 0.5% Triton lysis buffer and subjected to anti-HA or anti-Myc immunoprecipitation and further resolved on SDS-PAGE. The level of HA-Vpr, actin, exogenous and endogenous DCAF1, endogenous DDB1, Myc-DCAF1 WD and Myc-DCAF1 1377 were monitored in cell extracts as well as in the immunoprecipitated fractions by Western Blot using specific antibodies. * denotes the light chain of the IgG used for immunoprecipitation. # represents non-specific immunoprecipitated proteins. B. Cell cycle profile of transfected cells (GFP+) analyzed in A. G 2 /M:G 1 ratios were determined using the Modfit Software. C. The graph depicts the mean G 2 /M:G 1 ratios obtained in two independent experiments. Errors bars represent SEM.

Article Snippet: The mathematical model MODFIT (Verity Software House) was used to calculate the proportion of cells in the G 2 /M versus G 1 phase of the cell cycle.

Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, Lysis, Immunoprecipitation, SDS Page, Western Blot, Software