Fig. 8

Both sponge and human R-RAS2 homologs regulate focal adhesions in MDA-MB-231 cells. A Twenty-four hours upon transfection with either empty vector of vector containing EsuRRAS2-like-FLAG or HsaRRAS2-FLAG construct, focal adhesions were isolated and WB analysis of talin1, vinculin and integrin β5 was performed. Numbers below blots represent the relative expression of proteins compared to control (empty-vector) normalized against amidoblack staining of focal adhesion isolates. Densitometry was done using the ImageJ software (National Institutes of Health, USA). B Quantification of Western blot data presented in (A) together with two independently performed biological replicas. Histogram data are plotted as mean ± SD (n = 3) relative to expression in cells transfected with an empty-vector that was set as 1 (indicated by a dotted line). Data were analyzed by unpaired Student’s t-test. *p < 0.05. C Forty-eight hours after transfection with either an empty-vector or a vector containing EsuRRAS2-like-GFP or HsaRRAS2-GFP construct, cells were fixed with paraformaldehyde and incubated with Alexa-Flour 488 conjugated phalloidin for F-actin visualization, anti-integrin β5 antibody followed by Alexa-Fluor 647-conjugated antibody and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar—10 μm. Esu-sponge Eunapius subterraneus, Hsa-human