Fig 1: WNT‐5A activates Cdc42 in breast cancer cells. (A) MDA‐MB468 cells were stimulated with rWNT‐5A (0.4 μg/mL) for the indicated periods of time (0–4 h) and lysed. The lysate was either directly analyzed by Western blot for its content of total Cdc42 or α‐tubulin or used for GST‐PAK1 PBD pull down and subsequent analysis of Cdc42 (Cdc42‐GTP). (B) Quantifications of Cdc42‐GTP in rWNT‐5A stimulated MDA‐MB468 cells were carried out by calculating Integrated Density Values and normalizing it against total Cdc42 levels. (C) In order to confirm the specificity of the WNT‐5A‐induced Cdc42 activation, analysis of active Cdc42 (Cdc42‐GTP) was performed after stimulating the parental MDA‐MB468 cells with rWNT‐5A (0.4 μg/mL) in the absence of presence of sFRP1 (3.5 μg/mL). (D) Lysates of stably transfected MDA‐MB468‐5A were either directly analyzed by Western blot for content of total Cdc42 or α‐tubulin or used for GST‐PAK1 PBD pull down and subsequent analysis of Cdc42 (Cdc42‐GTP) and (E) Quantifications of Cdc42‐GTP in MDA‐MB468‐5A cells were carried out by calculating Integrated Density Values. (F) MDA‐MB231 cells were stimulated with rWNT‐5A (0.4 μg/mL) for 4 h and analyzed as in panels (A–F). Statistical comparisons between means were made with one‐way ANOVA (with Dunnett's Multiple Comparison test for post analysis; B), and Student's t‐test (C, E and F). All error bars represent standard error of the mean (n = 4). *p < 0.05; **p < 0.01.