Journal: The Journal of Cell Biology

Article Title: Induction of Cell Scattering by Expression of β1 Integrins in β1-Deficient Epithelial Cells Requires Activation of Members of the Rho Family of Gtpases and Downregulation of Cadherin and Catenin Function

doi:

Figure Lengend Snippet: Expression of β1 integrins in GE11 and GD25 cells activates RhoA and Rac1 but not Cdc42. Lysates of GE11 and GD25 cells expressing either the control vector alone, β1A, β1D, IL2R, or IL2R-β1A were incubated with GST-rhotekin fusion protein for the RhoA assay, or with GST-PAK fusion protein for the Rac1 and Cdc42 assays. The presence of active, GTP-bound RhoA, Rac1, or Cdc42 was analyzed by immunoblotting. Total amounts of RhoA, Rac1, and Cdc42 were determined in total cell lysates.

Article Snippet: The GST-rhotekin or GST-PAK precoupled to Sepharose-glutathione beads (Amersham Pharmacia Biotech) were used to precipitate GTP-bound RhoA, Rac1, or Cdc42 from cleared lysates of cells.

Techniques: Expressing, Plasmid Preparation, Incubation, Western Blot

Journal:

Article Title: Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

doi: 10.1111/j.1471-4159.2005.03252.x

Figure Lengend Snippet: ToxB induces apoptosis and elicits a selective caspase-mediated degradation of Rac1 GTPase in CGNs. (a) CGNs were incubated for 24 h in complete medium (containing serum and 25 mm KCl) in the presence of either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL). Following incubation, cells were fixed in paraformaldehyde and nuclei were stained with Hoechst dye. CGNs exposed to ToxB showed marked nuclear condensation and fragmentation characteristic of apoptosis. Scale bar, 10 μm. (b) Representative immunoblots of lysates obtained from CGNs incubated as described in (a). The blots shown were obtained from the BD PowerBlot™ (see Experimental procedures for details) using specific monoclonal antibodies against Rac1 (upper blots), RhoA (middle blots), or Cdc42 (lower blots). The immunoreactive Rho family GTPases are indicated by the arrows. Other protein bands apparent on the membranes represent adjacent lanes of the PowerBlot™ that were probed for distinct proteins and are shown to give an indication of the overall equality of protein loading. (c) The fold change in protein content induced by 24 h of ToxB treatment for each of the Rho family members blotted for in (b) is shown. Data represent the means ± range of duplicate PowerBlot™ comparisons for each GTPase. The dotted lines indicate no change (-1 to +1 fold). (d) CGNs were exposed for 8 h to ToxB in the absence or presence of either a pan-caspase inhibitor (Boc-Asp(OMe)-FMK; 200 μm) or zVAD-FMK; 200 μm) or a proteasome inhibitor (MG132; 10 μm). Following incubation, cell lysates were immunoblotted for Rac1.

Article Snippet: To detect GTP-bound Rac1, agarose beads bound to a GST-PAK-protein binding domain were added to the remaining supernatant according to the manufacturer’s instructions (Upstate Biotechnology).

Techniques: Incubation, Staining, Western Blot, Bioprocessing

Journal:

Article Title: Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

doi: 10.1111/j.1471-4159.2005.03252.x

Figure Lengend Snippet: Adenoviral expression of N17Rac1 induces apoptosis of CGNs: dominant-negative mutants of RhoA or Cdc42 do not cause significant cell death. (a) On day 4 in vitro, CGN cultures were infected with adenovirus expressing either GFP alone or in combination with dominant-negative N17Rac1. At 72 h post-infection, CGNs were fixed in paraformaldehyde and nuclei were stained with Hoechst dye. Images shown represent composites of two fields each for GFP or GFP/N17Rac1. Infected cells are indicated by the arrows. CGNs infected with adenovirus expressing GFP alone showed large intact nuclei characteristic of healthy cells (upper panels). In contrast, CGNs infected with adenovirus expressing N17Rac1 displayed marked chromatin condensation and fragmentation indicative of apoptotic cell death (lower panels). Note that non-infected cells in the N17Rac1 condition remained healthy. Scale bar, 10 μm. (b) Quantitation of CGN apoptosis in GFP-expressing cells following 72 h of infection with adenoviruses expressing either GFP alone or in combination with dominant-negative mutants of Rac1, Cdc42, or RhoA at a final titer of 50 infectious particles per cell. Using these conditions, each of the adenoviruses infected approximately 15-20% of the CGNs in the cultures. The results shown represent the means ± SEM for four experiments, each conducted on triplicate coverslips in which approximately 100 GFP-positive CGNs were counted per condition per coverslip. *Significantly different from adenoviral GFP alone (p < 0.01).

Article Snippet: To detect GTP-bound Rac1, agarose beads bound to a GST-PAK-protein binding domain were added to the remaining supernatant according to the manufacturer’s instructions (Upstate Biotechnology).

Techniques: Expressing, Dominant Negative Mutation, In Vitro, Infection, Staining, Quantitation Assay

Journal:

Article Title: Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

doi: 10.1111/j.1471-4159.2005.03252.x

Figure Lengend Snippet: Rac1 GTP-loading is maintained in CGNs by trophic support and integrin-mediated cell attachment. CGNs were incubated under either attached conditions (a) in trophic factor-deprived medium containing 5 mm KCl and lacking serum (5K-Ser) for up to 4 h, detached conditions (b) for up to 2 h in medium containing 25 mm KCl and serum (25K + Ser), or incubated with RAD or RGD peptides (c) for 8 h. Following incubation, CGNs were immediately lysed on ice into a high Mg2+ buffer. Cell debris was removed by brief centrifugation and a small aliquot of the resulting lysate was separated for quantitation of total Rac in each sample. The remaining cell lysates were incubated for 1 h with an excess of the protein binding domain of the Rac effector, PAK (PAK-PBD), bound to agarose beads. The pellet was washed three times with lysis buffer and samples were electrophoresed through 15% polyacrylamide gels. Proteins were transferred to PVDF membranes which were immunoblotted with a monoclonal antibody against Rac1 and an HRP-conjugated secondary antibody. Immunoreactive Rac1 protein was detected using standard ECL techniques. GTP-loaded Rac was brought down in the PAK-PBD precipitation (middle blots). The PAK-PBD is detected non-specifically by the secondary antibody and is shown as a control for equal loading (upper blots). The total Rac in each lysate is also shown as a loading control as no significant loss is detected in Rac protein under these conditions (bottom blots). The blots shown are representative of results obtained in three separate experiments. Att, attached control.

Article Snippet: To detect GTP-bound Rac1, agarose beads bound to a GST-PAK-protein binding domain were added to the remaining supernatant according to the manufacturer’s instructions (Upstate Biotechnology).

Techniques: Cell Attachment Assay, Incubation, Centrifugation, Quantitation Assay, Protein Binding, Lysis, Control