Journal: Molecular and Cellular Biology
Article Title: Wild-Type NRas and KRas Perform Distinct Functions during Transformation ▿
Figure Lengend Snippet: Effect of signaling components on the actin cytoskeleton, focal adhesions, and transformation. (A) Anchorage-independent growth of wild-type, N ras −/− , and K ras −/− MEFs following knockdown of Akt or Raf (shRNAs directed to the genes encoding Akt and Raf are indicated by shAkt and shRaf, respectively) or infected with empty vector (V) or vector directing the expression of an shRNA to green fluorescent protein (shGFP). Results are means ± standard deviations (SDs) of three independent experiments. Protein knockdown was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (B) Shown is FITC-phalloidin staining (green) for stress fibers, vinculin staining (red) for focal adhesions, and DAPI staining (blue) for nuclei in wild-type and N ras -deficient cells following knockdown of Akt or Cdc42. Bar, 10 μm. (C) Same experiment as in panel A, except cells of the indicated genotype following knockdown of small GTPases Rac, RhoA, or Cdc42 were analyzed (shRNAs directed to the genes encoding these Rho GTPases are denoted shRac, shRhoA, and shCdc42, respectively). Results are means ± SDs of three independent experiments. Depletion of proteins was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (D) Same experiment as in panel B, except wild-type and K ras -deficient cells following knockdown of Raf or RhoA were analyzed. (E) Immunoblotting of whole-cell lysates prepared from cells of the indicated genotype was performed with antibodies to phosphorylated GSK-3β and total GSK-3β, phosphorylated ERK1 and ERK2, and total ERK1 and ERK2. (F) In the indicated cells, the presence of activated, GTP-bound Rac, Cdc42, and RhoA was assayed as described in Materials and Methods. Whole-cell lysates were analyzed for total Rac, Cdc42, and RhoA as loading controls. Panels showing activated Rho GTPases are representative of three independent experiments, and loading controls are from the same lysates used for activation assays.
Article Snippet: Total cell lysates (40 μg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and immunoblotted with the following primary antibodies: anti-simian virus 40 (SV40) TAg (Pab 101), anti-KRas (F234), anti-HRas (F235), anti-NRas (F155), anti-Cdc42 (B-8), anti-RhoA (26C4), and anti-phospho-cofilin (sc-21867-R) were from Santa Cruz Biotechnology; anti-c-Raf (9422), anti-phospho-Akt (Ser473; 9271), anti-Akt (9272), anti-phospho-p44/42 MAPK (ERK1/2) (9101), anti-p44/42 MAPK (ERK1/2) (9102), anti-phospho-MEK1/2 (MEK1 and -2; 9121), anti-MEK1/2 (9122), anti-phosho-GSK-3β (9336S), anti-FAK (3285), and anti-PTEN (9552) were from Cell Signaling; anti-pan-Ras (Calbiochem), anti-Rac1 (clone 23A8; Upstate/Millipore), anti-GSK-3β (clone 4G; Upstate/Millipore), anti-FAK-Y397 (44-624G; BioSource), anti-Glu tubulin (Chemicon), and antitubulin (Sigma) were also used.
Techniques: Transformation Assay, Infection, Plasmid Preparation, Expressing, shRNA, Staining, Activation Assay