Journal: The Journal of Cell Biology
Article Title: Suppression of RhoG activity is mediated by a syndecan 4-synectin-RhoGDI1 complex and is reversed by PKC? in a Rac1 activation pathway
Figure Lengend Snippet: S4 mediates baseline RhoG activity and associates with synectin, RhoGDI1, and RhoG. (a) Murine pulmonary microvascular endothelial cells from WT and S4 knockout mice were cultured in 0.5% FBS/DME for 24 h before assay of RhoG activity. β-Actin and RhoGDI were used as loading controls. (right) Quantification from three experiments is shown. *, P = 0.009. Error bar indicates SEM. (b) A synthetic biotinylated peptide corresponding to the transmembrane domain and cytoplasmic tail of S4 was used to pull down proteins after conjugation to streptavidin beads. Pull-downs were immunoblotted and probed for synectin (top) and RhoGDI (bottom). − indicates unconjugated streptavidin beads. In both cases, the target proteins were pulled down only when incubated with the S4 tail peptide. (c) Coimmunoprecipitations were performed with antibodies against RhoGDI and synectin in three cell lines: WT RFPECs (left), RFPECs expressing the S4-FcR chimera (middle), and RFPECs expressing the S4-FcR (PDZ−) chimera (right). β-Actin was used as a loading control. IP, immunoprecipitation. (d) Glutathione beads conjugated with recombinant RhoGDI1 (right) or without the recombinant protein (left) were incubated with lysates of RFPECs for 12 h at 4°C. After washing, proteins were denatured by boiling and subjected to SDS-PAGE. The proteins were transferred to a membrane and probed for RhoG and synectin. (e) RFPECs were transfected with the S4-FcR chimera. Cells were incubated with FITC-conjugated human IgG to stain the chimera (left), washed twice with PBS, fixed, permeabilized, and stained with antibodies against RhoGDI1 and RhoG (middle and right, respectively). Arrows indicate regions of colocalization. Bars, 10 µm.
Article Snippet: Mouse monoclonal anti–β actin antibodies were obtained from BD.
Techniques: Activity Assay, Knock-Out, Mouse Assay, Cell Culture, Conjugation Assay, Incubation, Expressing, Immunoprecipitation, Recombinant, SDS Page, Transfection, Staining