Figure 1.

Bcr regulates RhoA activity in keratinocytes, but has a minor effect on Rac. (A) RhoA activity was analyzed in SCC9 cells upon Bcr KD with three different siRNAs (siBcr#1–3). G-LISA experiments indicate a decrease in RhoA activity upon Bcr KD with all three siRNA oligonucleotides. (B) G-LISA experiments indicated no change in global Rac1 activity upon Bcr KD in SCC9 cells (the observed change for siBcr#2 was not significant). Stimulation of SCC9 cells with 0.1 ng/µl EGF for 2 min was used as a positive control for this assay. (C) Control and Bcr KD SCC9 cells were subjected to calcium switch, followed by analysis of RhoA activity using traditional GST-RBD pull-downs. Fold-change values over control quantified by densitometry are noted below blots. (D) G-LISA experiments for RhoA activity were performed upon loss of Bcr in NHEKs at different time points after addition of 1.2 mM Ca²⁺-containing media. (E) RhoA activity was analyzed in NHEKs expressing GFP, CFP-tagged WT Bcr, or a GEF-dead Bcr mutant (NE/AA), after addition of high calcium media for 1 h. (F) RhoC activity was analyzed in control and Bcr KD NHEKs using a traditional GST-RBD pull-down. (G and H) G-LISA experiments for Rac1 or Cdc42 activity were performed upon Bcr KD in NHEKs. For all graphs, fold-change values from three independent samples are represented with error bars indicating SD. *, P < 0.05.