Journal:

Article Title: Raf-MEK-Erk Cascade in Anoikis Is Controlled by Rac1 and Cdc42 via Akt

doi: 10.1128/MCB.21.19.6706-6717.2001

Figure Lengend Snippet: Cytoplasmic retention of activated Erk inhibits RacN17- and Cdc42N17-induced apoptosis. (A) Adherent MEFs were transfected with myc epitope-tagged MKP3C/S. After 24 h, the cells were starved by culturing in medium containing 1% FCS for 24 h (panels a, c, e, and g) and then were stimulated with 20% serum for 10 min (panels f and h) or 2 h (panels b and d). Cells were stained with anti-ERK antibody (panels c and d) or anti-phospho-ERK antibody (panels g and h). The transfected cells (arrowheads) were identified by immunofluorescence detection of myc-MKP3C/S (panels a, b, e, and f). Bar, 10 μm. The results shown are representative of four independent experiments. (B) Adherent cells were cotransfected with the indicated constructs and truncated rat CD2 as a marker of transfection. Apoptosis in the transfected-cell population was assayed by the Annexin V binding assay. The results represent the means and SDs from two independent experiments performed in triplicate. (C) Western blot analysis of the levels of expression of the myc epitope-tagged GTPases with the different cotransfection combinations.

Article Snippet: The cells were then fixed in 3.7% formaldehyde (Sigma), rinsed with phosphate-buffered saline (PBS), incubated at room temperature for 2 h with biotinylated anti-rat CD2 antibody (OX-34; Cedarlane), rinsed with PBS, and incubated for 30 min with Streptavidin-Tri Color (Caltag).

Techniques: Transfection, Staining, Immunofluorescence, Construct, Marker, Binding Assay, Western Blot, Expressing, Cotransfection

Journal:

Article Title: Raf-MEK-Erk Cascade in Anoikis Is Controlled by Rac1 and Cdc42 via Akt

doi: 10.1128/MCB.21.19.6706-6717.2001

Figure Lengend Snippet: Dominant-negative Akt mutants lead to Erk activation. (A) MEFs were cotransfected with either of two Akt dominant-negative mutants, Akt K179M or Akt T308AS473A (AktAA), or an empty vector as a negative control. The cells were processed as described in the legend to Fig. ​Fig.3A.3A. (B) Cells were transfected with an HA-tagged Erk2 construct, a dominant-negative mutant of either Rac1 or Cdc42, or a myristoylated form of Akt (Akt-myr). At 24 h after transfection, cells were lysed, HA-Erk was immunoprecipitated, and its kinase activity was measured. (C) MEFs were cotransfected with a truncated rat CD2 antigen and either pcDNA3 as a negative control or a dominant-negative form of Rac1 or Cdc42 (RacN17 or CdcN17). A myristoylated form of Akt (Akt-myr) was also included where indicated. Cell survival was quantitated by Annexin V labeling of the transfected subpopulation identified by anti-CD2 staining. Results represent means and SDs for three experiments. (D) Cells were transfected with HA-tagged Erk2 and the HA-tagged Akt K179M mutant with or without m-Ksr1 (CA5). Erk2 kinase activity was assayed as described in the legend to Fig. ​Fig.33A.

Article Snippet: The cells were then fixed in 3.7% formaldehyde (Sigma), rinsed with phosphate-buffered saline (PBS), incubated at room temperature for 2 h with biotinylated anti-rat CD2 antibody (OX-34; Cedarlane), rinsed with PBS, and incubated for 30 min with Streptavidin-Tri Color (Caltag).

Techniques: Dominant Negative Mutation, Activation Assay, Plasmid Preparation, Negative Control, Transfection, Construct, Immunoprecipitation, Activity Assay, Labeling, Staining, Mutagenesis

Journal:

Article Title: Raf-MEK-Erk Cascade in Anoikis Is Controlled by Rac1 and Cdc42 via Akt

doi: 10.1128/MCB.21.19.6706-6717.2001

Figure Lengend Snippet: Inhibition of Akt, in conjunction with p53 activation, mimics apoptosis induction by dominant-negative forms of Rac1 and Cdc42. Exponentially growing MEFs were cotransfected with truncated rat CD2 and a combination of the following constructs, as indicated below the bars: a dominant-negative Cdc42 mutant (CdcN17), a dominant-negative Rac1 mutant (RacN17), wild-type p53 (p53), a kinase-dead Akt mutant (Akt179M), and an inactive Akt mutant (AktAA). Empty vector pcDNA3 was used as a control. Where indicated, the MEK inhibitor PD98059 (20 μM) was included in the culture medium. At 48 h after transfection, apoptotic cells in the transfected subpopulations were scored by Annexin V labeling. The data represent the means and SDs from three experiments.

Article Snippet: The cells were then fixed in 3.7% formaldehyde (Sigma), rinsed with phosphate-buffered saline (PBS), incubated at room temperature for 2 h with biotinylated anti-rat CD2 antibody (OX-34; Cedarlane), rinsed with PBS, and incubated for 30 min with Streptavidin-Tri Color (Caltag).

Techniques: Inhibition, Activation Assay, Dominant Negative Mutation, Construct, Mutagenesis, Plasmid Preparation, Transfection, Labeling